Methods and Compositions for the Diagnosis of Cancer Susceptibilities and Defective DNA Repair Mechanisms and Treatment Thereof

ABSTRACT

Methods and compositions for the diagnosis of cancer susceptibilities, defective DNA repair mechanisms and treatments thereof are provided. Among sequences provided here, the FANCD2 gene has been identified, and probes and primers are provided for screening patients in genetic-based tests and for diagnosing Fanconi Anemia and cancer. The FANCD2 gene can be targeted in vivo for preparing experimental mouse models for use in screening new therapeutic agents for treating conditions involving defective DNA repair. The FANCD2 polypeptide has been sequenced and has been shown to exist in two isoforms identified as FANCD2-S and the monoubiquinated FANCD-L form. Antibodies including polyclonal and monoclonal antibodies have been prepared that distinguish the two isoforms and have been used in diagnostic tests to determine whether a subject has an intact Fanconi Anemia/BRCA pathway.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/749,419, filed Mar. 29, 2010, pending, which is a continuation ofU.S. application Ser. No. 10/165,099, filed Jun. 6, 2002, abandoned,which is a continuation-in-part of U.S. application Ser. No. 09/998,027,filed Nov. 2, 2001, abandoned, which in turn claims priority from U.S.Provisional Application No. 60/245,756, filed Nov. 3, 2000. The entirecontents of each of the above applications are incorporated herein byreference.

GOVERNMENT SUPPORT

The work described herein was supported by the National Institute ofHealth, NIH Grant No. Health grants RO1HL52725-04, RO1DK43889-09,1PO1HL48546, and PO1HL54785-04. The US Government has certain rights tothe claimed invention.

INCORPORATION BY REFERENCE

The contents of the text file named “20363_(—)201C01US_ST25.txt”, whichwas created on Oct. 29, 2002 and is 132 KB in size, are herebyincorporated by reference in their entirety.

BACKGROUND

The present invention relates to the diagnosis of cancersusceptibilities in subjects having a defect in the FANCD2 gene and thedetermination of suitable treatment protocols for those subjects whohave developed cancer. Animal models with defects in the FANCD2 gene canbe used to screen for therapeutic agents.

Fanconi Anemia (FA) is an autosomal recessive cancer susceptibilitysyndrome characterized by birth defects, bone marrow failure and cancerpredisposition. Cells from FA patients display a characteristichypersensitivity to agents that produce interstrand DNA crosslinks suchas mitomycin C or diepoxybutane. FA patients develop several types ofcancers including acute myeloid leukemias and cancers of the skin,gastrointestinal; and gynecological systems. The skin andgastrointestinal tumors are usually squamous cell carcinomas. At least20% of patients with FA develop cancers. The average age of patients whodevelop cancer is 15 years for leukemia, 16 years for liver tumors and23 years for other tumors. (D'Andrea et al., Blood, (1997) Vol. 90, p.1725, Garcia-Higuera et al., Curr. Opin. Hematol., (1999) Vol. 2, pp.83-88 and Heijna et al., Am. J. Hum. Genet. Vol. 66, pp. 1540-1551).

FA is genetically heterogeneous. Somatic cell fusion studies haveidentified at least seven distinct complementation groups (Joenje etal., (1997) Am. J. Hum. Genet., Vol. 61, pp. 940-944 and Joenje et al.,(2000) Am. J. Hum. Genet, Vol. 67, pp. 759-762). This observation hasresulted in the hypothesis that the FA genes define a multicomponentpathway involved in cellular responses to DNA cross-links. Five of theFA genes (FANCA, FANCC, FANCE, FANCF and FANCG) have been cloned and theFANCA, FANCC and FANCG proteins have been shown to form a molecularcomplex with primarily nuclear localization. FANCC also localizes in thecytoplasm. Different FA proteins have few or no known sequence motifswith no strong homologs of the FANCA, FANCC, FANCE, FANCF, and FANCGproteins in non-vertebrate species. FANCF has weak homology of unknownsignificance to an E. Coli RNA binding protein. The two most frequentcomplementation groups are FA-A and FA-C which together account for75%-80% of FA patients. Multiple mutations have been recognized in theFANCA gene that span 80 kb and consists of at least 43 exons. FANCC hasbeen found to have 14 exons and spans approximately 80 kb. A number ofmutations in the FANCC gene have been identified which are correlatedwith FA of differing degrees of severity. FA-D has been identified as adistinct but rare complementation group. Although FA-D patients arephenotypically distinguishable from patients from other subtypes, the FAprotein complex assembles normally in FA-D cells (Yamashita et al.,(1998) P.N.A.S., Vol. 95, pp. 13085-13090).

The cloned FA proteins encode orphan proteins with no sequencesimilarity to each other or to other proteins in GenBank and nofunctional domains are apparent in the protein sequence. Little is knownregarding the cellular or biochemical function of these proteins.

Diagnosis of FA is complicated by the wide variability in FA patientphenotype. Further confounding diagnosis, approximately 33% of patientswith FA have no obvious congenital abnormalities. Moreover, existingdiagnostic tests do not differentiate FA carriers from the generalpopulation. The problems associated with diagnosis are described inD'Andrea et al., (1997). Many cellular phenotypes have been reported inFA cells but the most consistent is hypersensitivity to bifunctionalalkylating agents such as mitomycin C or diepoxybutane. These agentsproduce interstrand DNA cross-links (an important class of DNA damage).

Diagnosing cancer susceptibility is complicated because of the largenumber of regulatory genes and biochemical pathways that have beenimplicated in the formation of cancers. Different cancers depending onhow they arise and the genetic lesions involved may determine how asubject responds to any particular therapeutic treatments. Geneticlesions that are associated with defective repair mechanisms may giverise to defective cell division and apoptosis which in turn may increasea patient's susceptibility to cancer. FA is a disease condition in whichmultiple pathological outcomes are associated with defective repairmechanisms in addition to cancer susceptibility.

An understanding of the molecular genetics and cell biology of FanconiAnemia pathway can provide insights into prognosis, diagnosis andtreatment of particular classes of cancers and conditions relating todefects in DNA repair mechanisms that arise in non-FA patients as wellas FA patients.

SUMMARY OF THE INVENTION

The invention features a method of diagnosing or determining if apatient has cancer or is at increased risk of cancer, where the methodincludes testing a Fanconi Anemia/BRCA pathway gene for the presence ofa cancer-associated defect, where said presence of one or morecancer-associated defects is indicative of cancer or an increased riskof cancer in said patient. The cancer can be breast, ovarian, orprostate cancer, or other forms of cancer. The cancer-associated defectcan be one which results in a reduction in the ratio of FANC D2-Lrelative to FANC D2-S as compared to the ratio in a patient without oneor more cancer-associated defects in a Fanconi Anemia/BRCA pathway gene.

The invention also features a method of diagnosing or determining if apatient has cancer or is at increased risk of cancer, where the methodincludes testing a Fanconi Anemia/BRCA pathway protein for the presenceof a cancer-associated defect, where said presence of acancer-associated defect is indicative of cancer or an increased risk ofcancer in said patient. The cancer can be breast, ovarian, or prostatecancer, or other forms of cancer.

An another aspect, the invention features a method of diagnosing ordetermining if a patient is at increased risk of developing cancer,where the method includes the steps of (a) providing a tissue samplefrom said patient; (b) inducing DNA damage in the cells of said tissuesample; and (c) assaying for the presence of FANC D2-S and FANC D2-Lproteins in said cells; wherein a reduction in the ratio of FANC D2-L toFANC D2-S is indicative that said patient is at increased risk ofdeveloping cancer. The cancer can be breast, ovarian, or prostatecancer, or other forms of cancer. The patient can be known or not knownto have any previously-known cancer-associated defects in the BRCA-1 orBRCA-2 genes. A plurality of such tissue samples can be distributed onor in an array.

An another aspect, the invention features a method of determining if apatient has cancer, or is at increased risk of developing cancer, wherethe patient has no known cancer causing defect in the BRCA 1 or BRCA-2genes, where the method comprises the steps of: (a) providing a DNAsample from said patient; (b) amplifying the FANC D2 gene from saidpatient with the FANC D2 gene-specific polynucleotide primers of SEQ IDNOs:115-186; (c) sequencing the amplified FANC D2 gene; and (d)comparing the FANC D2 gene sequence from said patient to a referenceFANC D2 gene sequence, where a discrepancy between the two genesequences indicates the presence of a cancer-associated defect; wherethe presence of one or more cancer-associated defects indicates saidpatient has cancer or is at an increased risk of developing cancer. Thecancer can be breast, ovarian, or prostate cancer, or other forms ofcancer. The patient can be known or not known to have anypreviously-known cancer-associated defects in the BRCA-1 orFANC-D1/BRCA-2 genes. A plurality of such tissue samples can bedistributed on or in an array. SEQ ID NOs: 115-186 are matched sets ofprimers, as shown in Table 7, with the odd-numbered primers beingforward primers, and the even-numbered primers being reverse primers.Primers can also be used from different pairs, to make new pairings ofprimers, e.g., SEQ ID NO:115 can be used with SEQ ID NO:118, etc. By“discrepancy” is meant a difference between the two sequences, where thedifference is know to be associated with cancer.

In a further aspect, the invention features a method of screening for achemosensitizing agent, where the method comprises the steps of: (a)providing a potential inhibitor of the Fanconi Anemia/BRCA pathway; (b)providing a tumor cell line that is resistant to one or moreanti-neoplastic agents; (c) contacting said tumor cell line and saidpotential inhibitor of the Fanconi Anemia/BRCA pathway and said one ormore anti-neoplastic agents; and (d) measuring the growth rate of saidtumor cell line in the presence of said inhibitor of the FanconiAnemia/BRCA pathway and said anti-neoplastic agent; where a reducedgrowth rate of the tumor cell line, relative to cells of the tumor cellline in the presence of the anti-neoplastic agent and the absence ofsaid inhibitor of the Fanconi Anemia/BRCA pathway, is indicative thatthe potential inhibitor is a chemosensitizing agent. The potentialinhibitors of the Fanconi Anemia/BRCA pathway can be screened on amicroarray, where the microarray contains addresses containing one ormore cells that are resistant to one or more anti-neoplastic agents. Thepotential inhibitor of the Fanconi Anemia/BRCA pathway can be aninhibitor of the ubiquitination of the FANC D2 protein. Theanti-neoplastic agent can be cisplatin. The tumor cell line can be anovary cancer cell line.

In another aspect, the invention features a method of treating a patienthaving a cancer, where the cancer is resistant to a anti-neoplasticagent, where the method comprises the step of administering atherapeutically effective amount of an inhibitor of the FanconiAnemia/BRCA pathway together with said anti-neoplastic agent. Theanti-neoplastic agent can be cisplatin. The potential inhibitor of theFanconi Anemia/BRCA pathway can be an inhibitor of the ubiquitination ofthe FANC D2 protein. The tumor cell line can be an ovary cancer cellline.

In an additional aspect, the invetion features a method for screeningfor a cancer therapeutic, where the method comprises the steps of: (a)providing one or more cells containing a Fanconi Anemia/BRCA pathwaygene having one or more cancer associated defects; (b) growing saidcells in the presence of a potential cancer therapeutic; and (c)determining the rate of growth of said cells in the presence of saidpotential cancer therapeutic relative to the rate of growth ofequivalent cells grown in the absence of said potential cancertherapeutic; where a reduced rate of growth of said cells in thepresence of said potential cancer therapeutic, relative to the rate ofgrowth of equivalent cells grown in the absence of said potential cancertherapeutic, indicates that the potential cancer then is a cancertherapeutic. The cells can contain a Fanconi Anemia/BRCA pathway genehaving one or more cancer associated defects are distributed in a array,or several such genes.

The invention also features a method of predicting the efficacy of atherapeutic agent in a cancer patient, where the method comprises thesteps of: (a) providing a tissue sample from said cancer patient who isbeing treated with said therapeutic agent; (b) inducing DNA damage inthe cells of said tissue sample; and (c) detecting the presence of FANCD2-L protein in said cells; where the presence of FANC D2-L isindicative of a reduced efficacy of said therapeutic agent in saidcancer patient. The therapeutic agen can be an anti-neoplastic agent,e.g., can be cisplatin. Alternatively, in step (c), one can detect bothFANC-D2-S and FANC-D2-L, where a reduction in the ratio of FANC D2-Lrelative to FANC D2-S as compared to the ratio in a non-cancer patientindicates reduced efficacy.

The invention also features a method of determining resistance of tumorcells to an anti-neoplastic agent, comprising the steps of (a) providinga tissue sample from a patient who is being treated with ananti-neoplastic agent; (b) inducing DNA damage in the cells of saidtissue sample; and (c) determining the methylation state of a FanconiAnemia BRCA pathway gene; where methylation of a Fanconi Anemia/BRCAgene is indicative of resistance of the tumor cells to ananti-neoplastic agent. The Fanconi Anemia/BRCA gene can be the FANC Fgene. The anti-neoplastic agent can be cisplatin.

The invention also features a kit for detecting defects in the FANC D2gene, comprising a polynucleotide primer pair specific for the FANC D2gene, a reference FANC D2 gene sequence and packaging materialstherefore.

The invention also features a kit for detecting the presence of FANCD2-L, comprising a FANC D2-L-specific antibody and packaging materialstherefore.

The invention also features a kit for determining the methylation stateof a Fanconi Anemia/BRCA pathway gene, comprising FANC D2 polynucleotideprimer pairs and probes, a control unmethylated reference FANC D2 genesequence and packaging materials therefore.

The invention also features a kit for screening for a chemosensitizingagent, comprising a tumor cell line that is resistant to one or moreanti-neoplastic agents and packaging materials therefore. The tumor cellline can be an ovary tumor cell line, e.g., a cisplatin resistant ovarytumor cell line. The anti-neoplastic agent can be cisplatin.

The invention also features a microarray containing one or more nucleicacid sequences from one or more Fanconi Anemia/BRCA pathway genes. Thegenes can be on 15 selected from the group consisting of: ATM, FANC A,FANC B, FANC C, FANC D1, FANC D2, FANC E, FANC F and FANC G.

The invention also features the use of such a microarray in a method ofdetermining if a patient has cancer, or is at increased risk ofdeveloping cancer, where the method comprises the steps of (a) providingthe microarray; (b) providing a nucleic acid sample from said patient;(c) hybridizing said nucleic acid sample to said nucleic acid sequencesfrom the Fanconi Anemia/BRCA pathway on said microarray; and (d)detecting the presence of mutations in the Fanconi Anemia/BRCA pathwaygenes in the nucleic acid sample from said patient; where detecting thepresence of mutations is indicative of a patient who has cancer, or isat increased risk of developing cancer.

In a one embodiment of the invention there is provided an isolatednucleic acid molecule that includes a polynucleotide selected from (a) anucleotide sequence encoding a polypeptide having an amino acid sequenceas shown in SEQ ID NO:4; (b) a nucleotide sequence at least 90%identical to the polynucleotide of (b); (c) a nucleotide sequencecomplementary to the polynucleotide of (b); (d) a nucleotide sequence atleast 90% identical to the nucleotide sequence shown in SEQ ID NO:5-8,187-188; and (e) a nucleotide sequence complementary to the nucleotidesequence of (d). The polynucleotide may be an RNA molecule or a DNAmolecule, such as a cDNA.

In another embodiment of the invention, an isolated nucleic acidmolecule is provided that consists essentially of a nucleotide sequenceencoding a polypeptide having an amino acid sequence sufficientlysimilar to that of SEQ ID NO:4 to retain the biological property ofconversion from a short form to a long form of FANCD2 in the nucleus ofa cell for facilitating DNA repair. Alternately, the isolated nucleicacid molecule consists essentially of a polynucleotide having anucleotide sequence at least 90% identical to SEQ ID NO:9-191 orcomplementary to a nucleotide sequence that is at least 90% identical toSEQ ID NO:9-191.

In an embodiment, a method is provided for making a recombinant vectorthat includes inserting any of the isolated nucleic acid moleculesdescribed above into a vector. A recombinant vector product may be madeby this method and the vector may be introduced to form a recombinanthost cell into a host cell.

In an embodiment of the invention, a method is provided for making anFA-D2 cell line, that includes (a) obtaining cells from a subject havinga biallelic mutation in a complementation group associated with FA-D2;and (b) infecting the cells with a transforming virus to make the FA-D2cell line where the cells may be selected from fibroblasts andlymphocytes and the transforming virus selected from Epstein Barr virusand retrovirus. The FA-D2 cell line may be characterized by determiningthe presence of a defective FANDC2 in the cell line for example byperforming a diagnostic assay selected from (i) a Western blot ornuclear immunofluorescence using an antibody specific for FANCD2 and(ii) a DNA hybridization assay.

In an embodiment of the invention, a recombinant method is provided forproducing a polypeptide, that includes culturing a recombinant host cellwherein the host cell includes any of the isolated nucleic acidmolecules described above.

In an embodiment of the invention, an isolated polypeptide, including anamino acid sequence selected from (a) SEQ ID NO:4; (b) an amino acidsequence at least 90% identical to (a); (c) an amino acid sequence whichis encoded by a polynucleotide having a nucleotide sequence which is atleast 90% identical to at least one of SEQ ID NO:5-8, 187-188; (d) anamino acid sequence which is encoded by a polynucleotide having anucleotide sequence which is at least 90% identical to a complementarysequence to at least one of SEQ ID NO:5-8, 187-188; and (e) apolypeptide fragment of (a)-(d) wherein the fragment is at least 50aminoacids in length.

The isolated polypeptide may be encoded by a DNA having a mutationselected from nt 376A to G, nt 3707G to A, nt 904C to T and nt 958C toT. Alternatively, the polypeptide may be characterized by a polymorphismin DNA encoding the polypeptide, the polymorphism being selected from nt1122A to 0, nt 1440T to C, nt 1509C to T, nt 2141C to T, nt 2259T to C,nt 4098T to G, nt 4453G to A. Alternatively, the polypeptide may becharacterized by a mutation at amino acid 222 or amino acid 561.

In an embodiment of the invention, an antibody preparation is describedhaving a binding specificity for a FANCD2 protein where the antibody maybe a monoclonal antibody or a polyclonal antibody and wherein the FANCD2may be FANCD2-S or FANCD2-L.

In an embodiment of the invention, a diagnostic method is provided formeasuring FANCD2 isoforms in a biological sample where the methodincludes (a) exposing the sample to a first antibody for forming a firstcomplex with FANCD2-L and optionally a second antibody for forming asecond complex with FANCD2-S; and (b) detecting with a marker, theamount of the first complex and the second complex in the sample. Thesample may be intact cells or lysed cells in a lysate. The biologicalsample may be from a human subject with a susceptibility to cancer orhaving the initial stages of cancer. The sample may be from a cancer ina human subject, wherein the cancer is selected from melanoma, leukemia,astocytoma, glioblastoma, lymphoma, glioma, Hodgkins lymphoma, chroniclymphocyte leukemia and cancer of the pancreas, breast, thyroid, ovary,uterus, testis, pituitary, kidney, stomach, esophagus and rectum. Thebiological sample may be from a human fetus or from an adult human andmay be derived from any of a blood sample, a biopsy sample of tissuefrom the subject and a cell line. The biological sample may be derivedfrom heart, brain, placenta, liver, skeletal muscle, kidney, pancreas,spleen, thymus, prostate, testis, uterus, small intestine, colon,peripheral blood or lymphocytes. The marker may be a fluorescent marker,the fluorescent marker optionally conjugated to the FANCD2-L antibody, achemiluminescent marker optionally conjugated to the FANCD2-L antibodyand may bind the first and the second complex to a third antibodyconjugated to a substrate. Where the sample is a lysate, it may besubjected to a separation procedure to separate FANCD2 isoforms and theseparated isoforms may be identified by determining binding to the firstor the second FANCD2 antibody.

In an embodiment of the invention, a diagnostic test is provided foridentifying a defect in the Fanconi Anemia pathway in a cell populationfrom a subject, that includes selecting an antibody to FANCD2 proteinand determining whether the amount of an FAND2-L isoform is reduced inthe cell population compared with amounts, in a wild type cellpopulation; such that if the amount of the FANCD2-L protein is reduced,then determining whether an amount of any of FANCA, FANCB, FANCC,FANCD1, FANCE, FANCF or FANCG protein is altered in the cell populationcompared with the wild type so as to identify the defect in the FanconiAnemia pathway in the cell population. In one example, the amount of anisoform relies on a separation of the FANCD2-L and FANCD2-S isoformswhere the separation may be achieved by gel electrophoresis or by amigration binding banded test strip.

In an embodiment of the invention, a screening assay for identifying atherapeutic agent, is provided that includes selecting a cell populationin which FAND2-L is made in reduced amounts; exposing the cellpopulation to individual members of a library of candidate therapeuticmolecules; and identifying those individual member molecules that causethe amount of FANCD2-L to be increased in the cell population. In oneexample, the cell population is an in vitro cell population. In anotherexample, the cell population is an in vivo cell population, the in vivopopulation being within an experimental animal, the experimental animalhaving a mutant FANCD2 gene. In a further example, the experimentalanimal is a knock-out mouse in which the mouse FAND2 gene has beenreplaced by a human mutant FANCD2 gene. In another example, a chemicalcarcinogen is added to the cell population in which FANCD2 is made inreduced amounts, to determine if any member molecules can cause theamount of FANCD2-L to be increased so as to protect the cells form theharmful effects of the chemical carcinogen.

In an embodiment of the invention, an experimental animal model isprovided in which the animal FANCD2 gene has been removed and optionallyreplaced by any of the nucleic acid molecules described above.

In an embodiment of the invention, a method is provided for identifyingin a cell sample from a subject, a mutant FANCD2 nucleotide sequence ina suspected mutant FANCD2 allele which comprises comparing thenucleotide sequence of the suspected mutant FANCD2 allele with the wildtype FANCD2 nucleotide sequence wherein a difference between thesuspected mutant and the wild type sequence identifies a mutant FANCD2nucleotide sequence in the cell sample. In one example, the suspectedmutant allele is a germline allele. In another example, identificationof a mutant FANCD2 nucleotide sequence is diagnostic for apredisposition for a cancer in the subject or for an increased risk ofthe subject bearing an offspring with Fanconi Anemia. In anotherexample, the suspected mutant allele is a somatic allele in a tumor typeand identifying a mutant FANCD2 nucleotide sequence is diagnostic forthe tumor type. In another example, the nucleotide sequence of the wildtype and the suspected mutant FANCD2 nucleotide sequence is selectedfrom a gene, a mRNA and a cDNA made from a mRNA. In another example,comparing the polynucleotide sequence of the suspected mutant FANCD2allele with the wild type FANCD2 polynucleotide sequence, furtherincludes selecting a FANCD2 probe which specifically hybridizes to themutant FANCD2 nucleotide sequence, and detecting the presence of themutant sequence by hybridization with the probe. In another example,comparing the polynucleotide sequence of the suspected mutant FANCD2allele with the wild type FANCD2 polynucleotide sequence, furthercomprises amplifying all or part of the FANCD2 gene using a set ofprimers specific for wild type FANCD2 DNA to produce amplified FANCD2DNA and sequencing the FANCD2 DNA so as to identify the mutant sequence.In another example, where the mutant FANCD2 nucleotide sequence is agermline alteration in the FANCD2 allele of the human subject, thealteration is selected from the alterations set forth in Table 3 andwhere the mutant FANCD2 nucleotide sequence is a somatic alteration, inthe FANCD2 allele of the human subject, the alteration is selected fromthe alterations set forth in Table 3.

In an embodiment of the invention, a method is provided for diagnosing asusceptibility to cancer in a subject which comprises comparing thegermline sequence of the FANCD2 gene or the sequence of its mRNA in atissue sample from the subject with the germline sequence of the FANCD2gene or the sequence of its mRNA wherein an alteration in the germlinesequence of the FANCD2 gene or the sequence of its mRNA of the subjectindicates the susceptibility to the cancer. An alteration may bedetected in a regulatory region of the FANCD2 gene. An alteration in thegermline sequence may be determined by an assay selected from the groupconsisting of (a) observing shifts in electrophoretic mobility ofsingle-stranded DNA on non-denaturing polyacrylamide gels, (b)hybridizing a FANCD2 gene probe to genomic DNA isolated from the tissuesample, (c) hybridizing an allele-specific probe to genomic DNA of thetissue sample, (d) amplifying all or part of the FANCD2 gene from thetissue sample to produce an amplified sequence and sequencing theamplified sequence, (e) amplifying all or part of the FANCD2 gene fromthe tissue sample using primers for a specific FANCD2 mutant allele, (f)molecularly cloning all or part of the FANCD2 gene from the tissuesample to produce a cloned sequence and sequencing the cloned sequence,(g) identifying a mismatch between (i) a FANCD2 gene or a FANCD2 mRNAisolated from the tissue sample, and (ii) a nucleic acid probecomplementary to the human wild-type FANCD2 gene sequence, whenmolecules (i) and (ii) are hybridized to each other to form a duplex,(h) amplification of FANCD2 gene sequences in the tissue sample andhybridization of the amplified sequences to nucleic acid probes whichcomprise wild-type FANCD2 gene sequences, (i) amplification of FANCD2gene sequences in the tissue sample and hybridization of the amplifiedsequences to nucleic acid probes which comprise mutant FANCD2 genesequences, (j) screening for a deletion mutation in the tissue sample,(k) screening for a point mutation in the tissue sample, (l) screeningfor an insertion mutation in the tissue sample, and (m) in situhybridization of the FANCD2 gene of said tissue sample with nucleic acidprobes which comprise the FANCD2 gene.

In an embodiment of the invention, a method is provided for diagnosing asusceptibility for cancer in a subject, includes: (a) accessing geneticmaterial from the subject so as to determine defective DNA repair; (b)determining the presence of mutations in a set of genes, the setcomprising FAND2 and at least one of FANCA, FANCB, FANCC, FANCD1,FANCDE, FANDF, FANDG, BRACA1 and ATM; and (c) diagnosing susceptibilityfor cancer from the presence of mutations, in the set of genes.

In an embodiment of the invention, a method is provided for detecting amutation in a neoplastic lesion at the FANCD2 gene in a human subjectwhich includes: comparing the sequence of the FANCD2 gene or thesequence of its mRNA in a tissue sample from a lesion of the subjectwith the sequence of the wild-type FANCD2 gene or the sequence of itsmRNA, wherein an alteration in the sequence of the FANCD2 gene or thesequence of its mRNA of the subject indicates a mutation at the FANCD2gene of the neoplastic lesion. A therapeutic protocol may be providedfor treating the neoplastic lesion according to the mutation at theFANCD2 gene of the neoplastic lesion.

In an embodiment of the invention, a method is provided for confirmingthe lack of a FANCD2 mutation in a neoplastic lesion from a humansubject which comprises comparing the sequence of the FANCD2 gene or thesequence of its mRNA in a tissue sample from a lesion of said subjectwith the sequence of the wild-type FANCD2 gene or the sequence of itsRNA, wherein the presence of the wild-type sequence in the tissue sampleindicates the lack of a mutation at the FANCD2 gene.

In an embodiment of the invention, a method is provided for determininga therapeutic protocol for a subject having a cancer, that includes (a)determining if a deficiency in FANCD2-L occurs in a cell sample from thesubject by measuring FANCD2 isoforms using specific antibodies; (b) if adeficiency is detected in (a), then determining whether the deficiencyis a result of genetic defect in non-cancer cells; and (c) if (b) ispositive, reducing the use of a therapeutic protocol that causesincreased DNA damage so as to protect normal tissue in the subject andif (b) is negative, and the deficiency is contained within a geneticdefect in cancer cells only, then increasing the use of a therapeuticprotocol that causes increased DNA damage so as to adversely affect thecancer cells.

In an embodiment of the invention, a method of treating a FA pathwaydefect in a cell target is provided that includes: administering aneffective amount of FANCD2 protein or an exogenous nucleic acid to thetarget. The FA pathway defect may be a defective FANCD2 gene and theexogenous nucleic acid vector may further include introducing a vectoraccording to those described above. The vector may be selected from amutant herpes virus, a E1/E4 deleted recombinant adenovirus, a mutantretrovirus, the viral vector being defective in respect of a viral geneessential for production of infectious new virus particles. The vectormay be contained in a lipid micelle.

In an embodiment of the invention, a method is provided for treating apatient with a defective FANCD2 gene, that includes providing apolypeptide described in SEQ ID NO:4, for functionally correcting adefect arising from a condition arising from the defective FANCD2 gene.

In an embodiment of the invention, a cell based assay for detecting a FApathway defect is provided that includes obtaining a cell sample from asubject; exposing the cell sample to DNA damaging agents; and detectingwhether FANCD2-L is upregulated, the absence of upregulation beingindicative of the FA pathway defect. In the cell-based assay, amounts ofFANCD2 may be measured by an analysis technique selected from:immunoblotting for detecting nuclear foci; Western blots to detectamounts of FANCD2 isoforms and quantifying mRNA by hybridizing with DNAprobes.

In an embodiment of the invention, a kit is provided for use indetecting a cancer cell in a biological sample, that includes (a) primerpair which binds under high stringency conditions to a sequence in theFANCD2 gene, the primer pair being selected to specifically amplify analtered nucleic acid sequence described in Table 7; and containers foreach of the primers.

As used herein, the “Fanconi Anemia/BRCA pathway” or “Fanconi AnemiaPathway” refers to the genes within the 7 complementation groups (FA-Ato FA-G), the BRCA-1 gene and the ATM gene and their respective proteinsthat interact in a pathway referred herein as the Fanconi Anemia/BRCApathway and regulate the cellular response to DNA damage (see FIG. 22).

The genes of the Fanconi Anemia/BRCA pathway are:

-   -   1) FANC-A (e.g., Genbank Accession No.: NM_(—)000135)    -   2) FANC-B (not yet cloned)    -   3) FANC-C (e.g., Genbank Accession No.: NM_(—)000136)    -   4) FANC-D1/(e.g., Genbank Accession No.: U43746) BRCA-2    -   5) FANC-D2 (e.g., Genbank Accession No.: NM_(—)033084)    -   6) FANC-E (e.g., Genbank Accession No.: NM_(—)021922)    -   7) FANC-F (e.g., Genbank Accession No.: NM_(—)022725)    -   8) FANC-G (e.g., Genbank Accession No.: BC000032)    -   9) BRCA-1 (e.g., Genbank Accession No.: U14680)    -   10) ATM (e.g., Genbank Accession No.: U33841)

As used herein, “testing a Fanconi Anemia/BRCA pathway protein for thepresence of a cancer-associated defect” refers to the method ofdetermining if a protein encoded by a Fanconi Anemia/BRCA pathway gene,as defined herein, harbors a defect, as defined herein, that can causeor is associated with a cancer in a patient.

As used herein, the term “defect” refers to any alteration of a gene orprotein within the Fanconi Anemia/BRCA pathway, and/or proteins, withrespect to any unaltered gene or protein within the Fanconi Anemia/BRCApathway.

“Alteration” of a gene includes, but is not limited to: a) alteration ofthe DNA sequence itself, i.e., DNA mutations, deletions, insertions,substitutions; b) DNA modifications affecting the regulation of geneexpression such as regulatory region mutations, modification inassociated chromatin, modications of intron sequences affecting mRNAsplicing, modification affecting the methylation/demethylation state ofthe gene sequence; c) mRNA modications affecting protein translation ormRNA transport or mRNA splicing.

“Alteration” of a protein includes, but is not limited to, amino aciddeletions, insertions, substitutions; modification affecting proteinphosphorylation or glycosylation; modifications affecting proteintransport or localization; modifications affecting the ability to formprotein complexes with one or more associated proteins or changes in theamino acid sequence caused by changes in the DNA sequence encoding theamino acid.

As used herein, the term “increased risk” or “elevated risk” refers tothe greater incidence of cancer in those patients having altered FanconiAnemia/BRCA genes or proteins as compared to those patients withoutalterations in the Fanconi Anemia/BRCA pathway genes or proteins.“Increased risk” also refers to patients who are already diagnosed withcancer and may have an increased incidence of a different cancer form.According to the invention, “increased risk” of cancer refers tocancer-associated defects in a Fanconi Anemia/BRCA pathway gene thatcontributes to a 50%, preferably 90%, more preferably 99% or moreincrease in the probability of acquiring cancer relative to patients whodo not have a cancer-associated defect in a Fanconi Anemia/BRCA pathwaygene.

As used herein, an “inhibitor of the Fanconi Anemia/BRCA pathway”,according to the invention, refers to any compound that disrupts FANCD2-L protein function either directly or indirectly. Disruption of FANCD2-L protein function can be achieved either through disruption of anyof the other FANC proteins upstream of the FANC D2 protein within thepathway, inhibition of the ubiquitination of the FANC D2-S to the FANCD2-L isoform, inhibition of subsequent nuclear transport of the FANCD2-L protein or disrupton of the association of the FANC D2-L proteinwith the nuclear BRCA DNA repair protein complex. An “inhibitor”according to the invention can be nucleic acids (anti-sense RNA or DNAoligonucleotides), proteins (humanized antibodies), peptides or smallmolecule drugs that specifically bind to FANC D2-L and disrupt FANC D2-Lprotein function. In a most preferred embodiment, the inhibitor of theFanconi Anemia/BRCA pathway is a small molecule inhibitor of themono-ubiquitination of the FANC D2 protein.

As used herein, a “reduction in the ratio of FANC D2-L relative to FANCD2-S” refers to a decrease in the percentage of the total amount of FANCD2 protein that is in the FANC D2-L isoform. In a preferred embodiment,the total amount of FANC D2 protein that is in the FANC D2-L isoform isat most 25%, preferably 10%, more preferably 1% and most preferably 0%.Such a reduction indicates a defect in one or more genes or proteins ofthe Fanconi Anemia/BRCA pathway, as defined herein.

As used herein, “testing a Fanconi Anemia/BRCA pathway protein for thepresence of a cancer-associated defect” refers to the method ofdetermining if a protein encoded within the 7 complementation groups (A,B, C, D, E, F and G) that comprise the Fanconi Anemia/BRCA gene pathway,harbor a defect or other mutation, as defined herein, that can cause orcontribute to a cancer in a patient.

As used herein, the term “inducing DNA damage” refers to both chemicaland physical methods of damaging DNA. Chemicals that damage DNA include,but are not limited to, acids/bases and various mutagens, such asethidium bromide, acridine orange, as well as free radicals. Physicalmethods include, but are not limited to, ionizing radiation, such as Xrays and gamma rays, and ultraviolet (UV) radiation. Both methods of“inducing DNA damage” can result in DNA mutations that typicallyinclude, but are not limited to, single-strand breaks, double-strandbreaks, alterations of bases, insertions, deletions or the cross-linkingof DNA strands.

As used herein, the term “tissue biopsy” refers to a biologicalmaterial, which is isolated from a patient. The term “tissue”, as usedherein, is an aggregate of cells that perform a particular function inan organism and encompasses cell lines and other sources of cellularmaterial including, but not limited to, a biological fluid for example,blood, plasma, sputum, urine, cerebrospinal fluid, lavages, andleukophoresis samples.

As used herein, the term “amplifying”, when applied to a nucleic acidsequence, refers to a process whereby one or more copies of a particularnucleic acid sequence is generated from a template nucleic acid,preferably by the method of polymerase chain reaction (Mullis andFaloona, 1987, Methods Enzymol., 155:335). “Polymerase chain reaction”or “PCR” refers to an in vitro method for amplifying a specific nucleicacid template sequence. The PCR reaction involves a repetitive series oftemperature cycles and is typically performed in a volume of 50-100 μl.The reaction mix comprises dNTPs (each of the four deoxynucleotidesdATP, dCTP, dGTP, and dTTP), primers, buffers, DNA polymerase, andnucleic acid template. The PCR reaction comprises providing a set ofpolynucleotide primers wherein a first primer contains a sequencecomplementary to a region in one strand of the nucleic acid templatesequence and primes the synthesis of a complementary DNA strand, and asecond primer contains a sequence complementary to a region in a secondstrand of the target nucleic acid sequence and primes the synthesis of acomplementary DNA strand, and amplifying the nucleic acid templatesequence employing a nucleic acid polymerase as a template-dependentpolymerizing agent under conditions which are permissive for PCR cyclingsteps of (i) annealing of primers required for amplification to a targetnucleic acid sequence contained within the template sequence, (ii)extending the primers wherein the nucleic acid polymerase synthesizes aprimer extension product. “A set of polynucleotide primers” or “a set ofPCR primers” can comprise two, three, four or more primers.

Other methods of amplification include, but are not limited to, ligasechain reaction (LCR), polynucleotide-specific base amplification (NSBA),or any other method known in the art.

As used herein, the term “polynucleotide primer” refers to a DNA or RNAmolecule capable of hybridizing to a nucleic acid template and acting asa substrate for enzymatic synthesis under conditions in which synthesisof a primer extension product which is complementary to a nucleic acidtemplate is catalyzed to produce a primer extension product which iscomplementary to the target nucleic acid template. The conditions forinitiation and extension include the presence of four differentdeoxyribonucleoside triphosphates and a polymerization-inducing agentsuch as DNA polymerase or reverse transcriptase, in a suitable buffer(“buffer” includes substituents which are cofactors, or which affect pH,ionic strength, etc.) and at a suitable temperature. The primer ispreferably single-stranded for maximum efficiency in amplification.“Primers” useful in the present invention are generally between about 10and 35 nucleotides in length, preferably between about 15 and 30nucleotides in length, and most preferably between about 18 and 25nucleotides in length.

As defined herein, “a tumor” is a neoplasm that may either be malignantor non-malignant. Tumors of the same tissue type originate in the sametissue, and may be divided into different subtypes based on theirbiological characteristics.

As used herein, the term “cancer” refers to a malignant disease causedor characterized by the proliferation of cells which have lostsusceptibility to normal growth control. “Malignant disease” refers to adisease caused by cells that have gained the ability to invade eitherthe tissue of origin or to travel to sites removed from the tissue oforigin.

As used herein, the term “antibody” refers to an immunoglobulin havingthe capacity to specifically bind a given antigen. The term “antibody”as used herein is intended to include whole antibodies of any isotype(IgG, IgA, IgM, IgE, etc), and fragments thereof which are alsospecifically reactive with a vertebrate, e.g., mammalian, protein.Antibodies can be fragmented using conventional techniques and thefragments screened for utility in the same manner as whole antibodies.Thus, the term includes segments of proteolytically-cleaved orrecombinantly-prepared portions of an antibody molecule that are capableof selectively reacting with a certain protein. Non-limiting examples ofsuch proteolytic and/or recombinant fragments include Fab, F(ab′)2, Fab,Fv, and single chain antibodies (scFv) containing a V[L] and/or V[H]domain joined by a peptide linker. The scFv's may be covalently ornon-covalently linked to form antibodies having two or more bindingsites. Antibodies may be labeled with detectable moieties by one ofskill in the art. In some embodiments, the antibody that binds to anentity one wishes to measure (the primary antibody) is not labeled, butis instead detected by binding of a labeled secondary antibody thatspecifically binds to the primary antibody.

A patient is “treated” according to the invention if one or preferablymore symptoms of cancer as described herein are eliminated or reduced inseverity, or prevented from progressing or developing further.

As used herein, the term “therapeutically effective amount” means thetotal amount of each active component of the pharmaceutical compositionor method that is sufficient to show a meaningful patient benefit, i.e.,treatment, healing, prevention or amelioration of the relevant medicalcondition, or an increase in rate of treatment, healing, prevention oramelioration of such conditions.

As used herein, the term “cancer therapeutic” refers to a compound thatprevents the onset or progression of cancer or prevents cancermetastasis or reduces, delays, or eliminates the symptoms of cancer.

As used herein, the term “inhibitor of the mono-ubiquitination” refersto a compound that prevents or inhibits the ubiquitination of the FANCD2 gene. “Ubiquitination” is defined as the covalent linkage ofubiquitin to a protein by a E3 mono-ubiquitin ligase. In a preferredembodiment, the “inhibitor of the mono-ubiquitination” refers to anyinhibitor of a FANC protein complex with E3 FANC D2 monoubiquitin ligaseactivity such that FANC D2 monoubiquitin ligase activity is inhibited.

As used herein, the term “cisplatin” refers to an agent with thefollowing chemical structure:

Cisplatin, also called cis-diamminedichloroplatinum(II), is one of themost frequently used anticancer drugs. It is an effective component ofseveral different combination drug protocols used to treat a variety ofsolid tumors. These drugs are used in the treatment of testicular cancer(with bleomycin and vinblastine), bladder cancer, head and neck cancer(with bleomycin and fluorouracil), ovarian cancer (with cyclophosphamideor doxorubicin) and lung cancer (with etoposide). Cisplatin has beenfound to be the most active single agent against most of these tumors.Cisplatin is commercially available as ‘Platinol’ from Bristol MyersSquibb Co. Cisplatin, is one of a number of platinum coordinationcomplexes with antitumor activity. The platinum compounds are DNAcross-linking agents similar to but not identical to the alkylatingagents. The platinum compounds exchange chloride ions for nucleophilicgroups of various kinds. Both the cis and trans isomers do this but thetrans isomer is known to be bioligically inactive for reasons notcompletely understood. To possess antitumor activity a platinum compoundmust have two relatively labile cis-oriented leaving groups. Theprincipal sites of reaction are the N7 atoms of guanine and adenine. Themain interaction is formation of intrastrand cross links between thedrug and neighboring guanines. Intrastrand cross linking has been shownto correlate with clinical response to cisplatin therapy. DNA/proteincross linking also occurs but this does not correlate with cytotoxicity.Cross-resistance between the two groups of drugs is usually not seenindicating that the mechanisms of action are not identical. The types ofcross linking with DNA may differ between the platinum compounds and thetypical alkylating agents.

As used herein, “resistance to one or more anti-neoplastic agents”refers the ability of cancer cells to develop resistance to anticancerdrugs. Mechanisms of drug resistance include decreased intracellulardrug levels caused by an increased drug efflux or decreased inwardtransport, increased drug inactivation, decreased conversion of drug toan active form, altered amount of target enzyme or receptor (geneamplification), decreased affinity of target enzyme or receptor fordrug, enhanced repair of the drug-induced defect, decreased activity ofan enzyme required for the killing effect (topoisomerase II). In apreferred embodiment of the invention, drug resistance refers to theenhanced repair of DNA damage induced by one or more anti-neoplasticagents. In another preferred embodiment of the invention, the enhancedrepair of DNA damage induced by one or more anti-neoplastic agents isdue to a constitutively active Fanconi Anemia/BRCA DNA repair pathway.

As used herein, the term “anti-neoplastic agent” refers to a compoundthat is used to treat cancer. According to the invention, an“anti-neoplastic agent” encompasses chemotherapy compounds as well asother anti-cancer agents known in the art. In a preferred embodiment,the “anti-neoplastic agent” is cisplatin. Anti-neoplastic agentsaccording to the invention also include cancer therapy protocols usingchemotherapy compounds in conjunction with radiation therapy and/orsurgery. Radiation therapy relies on the local destruction of cancercells through ionizing radiation that disrupts cellular DNA. Radiationtherapy can be externally or internally originated, high or low dose,and delivered with computer-assisted accuracy to the site of the tumor.Brachytherapy, or interstitial radiation therapy, places the source ofradiation directly into the tumor as implanted “seeds.”

As used herein, the term “a reduced growth rate” refers to a decrease of50%, preferably 90%, more preferably 99% and most preferably 100% in therate of cellular proliferation of a tumor cell line that is beingtreated with a potential inhibitor of the Fanconi Anemia/BRCA pathwayand one or more chemotherapy compounds relative to cells of a tumor cellline that is not being treated with a potential inhibitor of the FanconiAnemia/BRCA pathway and one or more chemotherapy compounds.

As used herein, the term “chemosensitizing agent” refers to any compoundthat renders a cell or cell population sensitive to a chemotherapycompound and results in a “reduced growth rate” as defined herein. Achemosensitizing agent is a compound that is generally not cytotoxic initself, but modifies the host or tumor cells to enhance anticancertherapy. According to the invention, cellular resistance to achemotherapy compound is reversed in the presence of a chemosensitizingagent. In a preferred embodiment, the chemosensitizing agent is aninhibitor of the Fanconi Anemia/BRCA pathway. In a most preferredembodiment, the chemosensitizing agent is an inhibitor of themono-ubiquitination of the FANC D2 protein.

As used herein, the “methylation state of a Fanconi Anemia/BRCA pathwaygene” refers to the presence of one or more methylated cytosines (5m-C)within a Fanconi Anemia/BRCA pathway gene and results in a decrease orinhibition of gene expression of 90%, 99% or preferably 100% relative toa gene that is not methylated. In a preferred embodiment, the methylatedcytosines reside within CpG islands. According to the invention, a geneis said to be “methylated” when one or more of CpG residues ismethylated.

As used herein, “microarray”, or “array”, refers to a plurality ofunique biomolecules attached to one surface of a solid support.Preferably, a biomolecule of the invention a potential inhibitor of theFanconi Anemia/BRCA pathway as described herein. In this embodiment, themicroarray of the invention comprises nucleic acids, proteins,polypeptides, peptides, fusion proteins or small molecules that areimmobilised on a solid support, generally at high density. Each of thebiomolecules is attached to the surface of the solid support in apre-selected region. Suitable solid supports are available commercially,and will be apparent to the skilled person. The supports may bemanufactured from materials such as glass, ceramics, silica and silicon.The supports usually comprise a flat (planar) surface, or at least anarray in which the molecules to be interrogated are in the same plane.In one embodiment, the array is on microbeads. In one embodiment, thearray comprises at least 10, 500, 1000, 10,000 different biomoleculesattached to one surface of the solid support.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing features of the invention will be more readily understoodby reference to the following detailed description, taken with referenceto the accompanying drawings, in which:

FIG. 1A provides a Western blot demonstrating that the Fanconi Anemiaprotein complex is required for the monoubiquitination of FANCD2. Normal(WT) cells (lane 1) express two isoforms of the FANCD2 protein, a lowmolecular weight isoform (FANCD2-S) (155 kD) and a high molecular weightisoform (FANCD2-L) (162 kD). Lanes 3, 7, 9, 11 show that FA cell linesderived from type A, C, G, and F patients only express the FANCD2-Sisoform. Lanes 4, 8, 10, 12 show the restoration of the high molecularweight isoform FANCD2-L following transfection of cell lines withcorresponding FAcDNA.

FIG. 1B shows a Western blot obtained after HeLa cells were transfectedwith a cDNA encoding HA-ubiquitin. After transfection, cells weretreated with the indicated dose of mitomycin C (MMC). Cellular proteinswere immunoprecipitated with a polyclonal antibody (E35) to FANCD2, asindicated. FANCD2 was immunoprecipitated, and immune complexes wereblotted with anti-FANCD2 or anti-HA monoclonal antibody.

FIG. 1C shows a Western blot obtained after HeLa cells were transfectedwith a cDNA encoding HA-ubiquitin. After transfection, cells weretreated with the indicated dose of ionizing radiation (IR). FANCD2 wasimmunoprecipitated, and immune complexes were blotted with anti-FANCD2or anti-HA monoclonal antibody.

FIG. 1D shows a Western blot obtained after PA-G fibroblast line(FAG326SV) or corrected cells (FAG326SV plus FANCG cDNA) weretransfected with the HA-Ub cDNA, FANCD2 was immunoprecipitated, andimmune complexes were blotted with anti-FANCD2 or anti-HA antisera.

FIG. 1E shows a Western blot obtained after treatment of HeLa cells with1 mM hydroxyurea for 24 hours. HeLa cell lysates were extracted andincubated at the indicated temperature for the indicated time periodwith or without 2.5 μM ubiquitin aldehyde. The FANCD2 protein wasdetected by immunoblot with monoclonal anti-FANCD2 (F117).

FIG. 2 demonstrates that the Fanconi Anemia pathway is required for theformation of FANCD2 nuclear foci. Top panel shows anti-FANCD2immunoblots of SV40 transformed fibroblasts prepared as whole cellextracts. Panels a-h show immunofluorescence with the affinity-purifiedanti-FANCD2 antiserum. The uncorrected (mutant, M) FA fibroblasts wereFA-A (GM6914), FA-G (FAG326SV), FA-C(PD426), and FA-D (PD20F). The FA-A,FA-G, and FA-C fibroblasts were functionally complemented with thecorresponding FA cDNA. The FA-D cells were complemented withneomycin-tagged human chromosome 3p (Whitney et al., 1995).

FIG. 3 shows the cell cycle dependent expression of the two isoforms ofthe FANCD2 protein. (a) HeLa cells, SV40 transformed fibroblasts from anFA-A patient (GM6914), and GM6914 cells corrected with FANCA cDNA weresynchronized by the double thymidine block method. Cells correspondingto the indicated phase of the cell cycle were lysed, and processed forFANCD2 immunoblotting (b) Synchrony by nocodazole block (c) Synchrony bymimosine block (d) HeLa cells were synchronized in the cell cycle usingnocodazole or (e) mimosine, and cells corresponding to the indicatedphase of the cell cycle were immunostained with the anti-FANCD2 antibodyand analyzed by immunofluorescence.

FIG. 4 shows the formation of activated FANCD2 nuclear foci followingcellular exposure to MMC, Ionizing Radiation, or Ultraviolet Light.Exponentially-growing HeLa cells were either untreated or exposed to theindicated DNA damaging agents, (a) Mitomycin C (MMC), (b) γ-irradiation(IR), or (c) Ultraviolet Light (UV), and processed for FANCD2immunoblotting or FANCD2 immunostaining. (a) Cells were continuouslyexposed to 40 ng/ml MMC for 0-72 hours as indicated, or treated for 24hours and fixed for immunofluorescence. (b) and (c) Cells were exposedto γ-irradiation (10 Gy, B) or UV light (60 J/m2 C) and collected afterthe indicated time (upper panels) or irradiated with the indicated dosesand harvested one hour later (lower panels). For immunofluorescenceanalysis cells were fixed 8 hours after treatment (B, 10 Gy, C, 60J/m2). (d) The indicated EBV-transformed lymphoblast lines from a normalindividual (PD7) or from various Fanconi Anemia patients were eithertreated with 40 ng/ml of Mitomycin C continuously (lanes 1-21) orexposed to 15 Gy of γ-irradiation (lanes 22-33) and processed for FANCD2immunoblotting. The upregulation of FANCD-L after MMC or IR treatmentwas seen in PD7 (lanes 2-5) and in the corrected FA-A cells (lanes28-33), but was not observed in any of the mutant Fanconi Anemia celllines. Similarly, IR-induced FANCD2 nuclear foci were not detected in PAfibroblasts (FA-G+IR) but were restored after functional complementation(PA-G+FANCG).

FIG. 5 shows co-localization of activated FANCD2 and BRCA1 in DiscreteNuclear Foci following DNA damage. HeLa cells were untreated or exposedto Ionizing Radiation (10 Gy) as indicated, and fixed 8 hours later. (a)Cells were double-stained with the D-9 monoclonal anti-BRCA1 antibody(green, panels a, d, g, h) and the rabbit polyclonal anti-FANCD2antibody (red, panels b, e, h, k), and stained cells were analyzed byimmunofluorescence. Where green and red signals overlap (Merge, panelsc, f, i, l) a yellow pattern is seen, indicating co-localization ofBRCA1 and FANCD2. (b) Co-immunoprecipitation of FANCD2 and BRCA1. HeLacells were untreated (IR) or exposed to 15 Gy of γ-irradiation (+IR) andcollected 12 hours later. Cell lysates were prepared, and cellularproteins were immunoprecipitated with either the monoclonal FANCD2antibody (FI-17, lanes 9-10), or any one of three independently-derivedmonoclonal antibodies to human BRCA1 (lanes 3-8): D-9 (Santa Cruz), Ab-1and Ab-3 (Oncogene Research Products). The same amount of purified mouseIgG (Sigma) was used in control samples (lanes 1-2). Immune complexeswere resolved by SDS-PAGE and were immunoblotted with anti-FANCD2 oranti-BRCA1 antisera. The FANCD-L isoform preferentiallycoimmunoprecipitated with BRCA1.

FIG. 6 shows the co-localization of activated FANCD2 and BRCA1 indiscrete nuclear foci during S phase. (a) HeLa cells were synchronizedin late G1 with mimosine and released into S phase. S phase cells weredouble-stained with the monoclonal anti-BRCA1 antibody (green, panels a,d) and the rabbit polyclonal anti-FANCD2 antibody (red, panels b, e),and stained cells were analyzed by immunofluorescence. Where green andred signals overlap (merge, panels c, f), a yellow pattern is seen,indicating co-localization of BRCA1 and FANCD2. (b) HeLa cellssynchronized in S phase were either untreated (a, b, k, l) or exposed toIR (50 Gy, panels c, d, m, n), MMC (20 μg/ml, panels c, f, o, p), or UV(100 j/m2, panels g, h, q, r) as indicated and fixed 1 hour later. Cellswere subsequently immunostained with an antibody specific for FANCD2 orBRCA1.

FIG. 7 shows that FANCD2 forms foci on synaptonemal complexes that canco-localize with BRCA1 during meiosis I in mouse spermatocytes. (a)Anti-SCP3 (white) and anti-FANCD2 (red) staining of synaptonemalcomplexes in a late pachytene mouse nucleus. (b) SCP3 staining of latepachytene chromosomes. (c) Staining of this spread with preimmune serumfor the anti-FANCD2 E35 antibody. (d) Anti-SCP3 staining of synaptonemalcomplexes in a mouse diplotene nucleus. (e) Costaining of this spreadwith E35 anti-FANCD2 antibody. Note staining of both the unpaired sexchromosomes and the telomeres of the autosomes with anti-FANCD2. (f)Costaining of this spread with anti-BRCA1 antibody. The sex chromosomesare preferentially stained. (g) Anti-FANCD2 staining of late pachytenesex chromosome synaptonemal complexes. (h) Anti-BRCA1 staining of thesame complexes. (i) Anti-FANCD2 (red) and anti-BRCA1 (green) co-staining(co-localization reflected by yellow areas).

FIG. 8 provides a schematic interaction of the FA proteins in a cellularpathway. The FA proteins (A, C, and G) bind in a functional nuclearcomplex. Upon activation of this complex, by either S phase entry or DNAdamage, this complex enzymatically modifies (monoubiquitinates) the Dprotein. According to this model, the activated D protein issubsequently targeted to nuclear foci where it interacts with the BRCA1protein and other proteins involved in DNA repair.

FIG. 9 shows a Northern blot of cells from heart, brain, placenta,liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate,testis, uterus, small intestine, colon and peripheral blood lymphocytesfrom a human adult and brain, lung, liver and kidney from a human fetusprobed with a full-length FANCD2 cDNA and exposed for 24 hours.

FIG. 10 shows allele specific assays for mutation analysis of 2 FANCD2families where the family pedigrees (a, d) and panels b, c, e and f arevertically aligned such that the corresponding mutation analysis isbelow the individual in question. Panels a-c depict the PD20 and panelsd-f the VU008 family. Panels b and e show the segregation of thematernal mutations as detected by the creation of a new MspI site (PD20)or DdeI site (VU008). The paternally inherited mutations in bothfamilies were detected with allele specific oligonucleotidehybridization (panels c and f).

FIG. 11 shows a Western blot analysis of the FANCD2 protein in humanFanconi Anemia cell lines. Whole cell lysates were generated from theindicated fibroblast and lymphoblast lines. Protein lysates (70 g) wereprobed directly by immunoblotting with the anti-FANCD2 antiserum. TheFANCD2 proteins (155 kD and 162 kD) are indicated by arrows. Other bandsin the immunoblot are non-specific. (a) Cell lines tested includedwild-type cells (lanes 1,7), PD20 Fibroblasts (lane 2), PD20lymphoblasts (lane 4), revertant MMC-resistant PD20 lymphoblasts (lane5, 6), and chromosome 3p complemented PD20 fibroblasts (lane 3). Severalother FA group D cell lines were analyzed including HSC62 (lane 8) andVU008 (lane 9). FA-A cells were HSC72 (lane 10), FA-C cells were PD4(lane 11), and FA-G cells were EUFA316 (lane 12). (b) Identification ofa third FANCD2 patient. FANCD2 protein was readily detectable inwild-type and FA group G cells but not in PD733 cells. (c) Specificityof the antibody. PD20i cells transduced with a retroviral FANCD2expression vector displayed both isoforms of the FANCD2 protein (lane 4)in contrast to empty vector controls (lane 3) and untransfected PD20icells (lane 2). In wild-type cells the endogenous FANCD2 protein (twoisoforms) was also immunoreactive with the antibody (lane 1).

FIG. 12 shows functional complementation of FA-D2 cells with the clonedFANCD2 cDNA. The SV40-transformed FA-D2 fibroblast line, PD20i, wastransduced with pMMP-puro (PD20+vector) or pMMP-FANCD2 (PD20+FANCD2 wt).Puromycin-selected cells were subjected to MMC sensitivity analysis.Cells analyzed were the parental PD20F cells (Δ), PD20 corrected withhuman chromosome 3p (◯), and PD20 cells transduced with either pMMP-puro

or pMMP-FANCD2(wt)-puro (♦).

FIG. 13 shows a molecular basis for the reversion of PD20 Lymphoblasts.(a) PCR primers to exons 5 and 6 were used to amplify cDNA. Controlsamples (right lane) yielded a single band of 114 bp, whereas PD20 cDNA(left lane) showed 2 bands, the larger reflecting the insertion of 13 bpof intronic sequence into the maternal allele. Reverted, MMC resistantlymphoblasts (middle lane) from PD20 revealed a third, inframe splicevariant of 114+36 bp (b) Schematic representation of splicing at theFANCD2 exon 5/intron 5 boundary. In wild-type cDNA 100% of splice eventsoccur at the proper exon/intron boundary (SEQ ID NO:189), whereas thematernal A->G mutation (indicated by arrow) leads to aberrant splicing,also in 100% (SEQ ID NO:190). In the reverted cells all cDNAs with thematernal mutation also had a second sequence change (fat arrow) andshowed a mixed splicing pattern with insertion of either 13 bp (˜40% ofmRNA) or 36 bp (˜60% of mRNA) (SEQ ID NO:191).

FIG. 14 shows an FANCD2 Western blot of cancer cell lines derived frompatients with ovarian cancer.

FIG. 15 shows a sequence listing for amino acid sequence of human FANCD2(SEQ ID NO:1) and alignment with fly (SEQ ID NO:2) and plant (SEQ IDNO:3) homologues using the BEAUTY algorithm (Worley et al., (1995)Genome Res. Vol. 5, pp. 173-184). Black boxes indicate amino acididentity and gray similarity. The best alignment scores were observedwith hypothetical proteins in D melanogaster (p=8.4×10-58, accessionnumber AAF55806) and A thaliana (p=9.4×10-45, accession number B71413).

FIG. 16 is the FANCD cDNA sequence -63 to 5127 nucleotides (SEQ ID NO:5)and polypeptide encoded by this sequence from amino acid 1 to 1472 (SEQID NO:4).

FIG. 17 is the nucleotide sequence for FANCD-S.ORF (SEQ ID NO:187)compared with FANCD cDNA (SEQ ID NO:188).

FIG. 18 is the nucleotide sequence for human FANCD2-L (SEQ ID NO:6).

FIG. 19 is the nucleotide sequence for human FANCD2-S (SEQ ID NO:7).

FIG. 20 is the nucleotide sequence for mouse FANCD2 (SEQ ID NO:8).

FIG. 21 depicts protocol used to analyze the methylation state of theFANC F gene.

FIG. 22 depicts the Fanconi Anemia/BRCA pathway.

DETAILED DESCRIPTION

“FANCD2-L therapeutic agent” shall mean any of a protein isoform, andincludes a peptide, a peptide derivative, analogue or isomer of theFANCD2-L protein and further include any of a small molecule derivative,analog, isomer or agonist that is functionally equivalent to FANCD2-L.Also included in the definition is a nucleic acid encoding FANCD2 whichmay be a full length or partial length gene sequence or cDNA or may be agene activating nucleic acid or a nucleic acid binding moleculeincluding an aptamer of antisense molecule which may act to modulategene expression.

“Nucleic acid encoding FANCD-2” shall include the complete cDNA orgenomic sequence of FANCD2 or portions thereof for expressing FANCD2-Lprotein as defined above. The nucleic acid may further be included in anucleic acid carrier or vector and includes nucleic acid that has beensuitably modified for effective delivery to the target site.

“Stringent conditions of hybridization” will generally includetemperatures in excess of 30° C., typically in excess of 37° C., andpreferably in excess of 45° C. Stringent salt conditions will ordinarilybe less than 1000 mM, typically less than 500 mM, and preferably lessthan 200 mM.

“Substantial homology or similarity” for a nucleic acid is when anucleic acid or fragment thereof is “substantially homologous” (or“substantially similar”) to another if, when optimally aligned (withappropriate nucleotide insertions or deletions) with the other nucleicacid (or its complementary strand), there is nucleotide sequenceidentity in at least about 60% of the nucleotide bases, usually at leastabout 70%, more usually at least about 80.

“Antibodies” includes polyclonal and/or monoclonal antibodies andfragments thereof including single chain antibodies and including singlechain antibodies and Fab fragments, and immunologic binding equivalentsthereof, which have a binding specificity sufficient to differentiateisoforms of a protein. These antibodies will be useful in assays as wellas pharmaceuticals.

“Isolated” is used to describe a protein, polypeptide or nucleic acidwhich has been separated from components which accompany it in itsnatural state. An “isolated” protein or nucleic acid is substantiallypure when at least about 60 to 75% of a sample exhibits a single aminoacid or nucleotide sequence.

“Regulatory sequences” refers to those sequences normally within 100 kbof the coding region of a locus, but they may also be more distant fromthe coding region, which affect the expression of the gene (includingtranscription of the gene, and translation, splicing, stability or thelike of the messenger RNA).

“Polynucleotide” includes RNA, cDNA, genomic DNA, synthetic forms, andmixed polymers, both sense and antisense strands, and may be chemicallyor biochemically modified or may contain non-natural or derivatizednucleotide bases, as will be readily appreciated by those skilled in theart. Such modifications include, for example, labels, methylation,substitution of one or more of the naturally occurring nucleotides withan analog, internucleotide modifications such as uncharged linkages(e.g., methyl phosphonates, phosphotriesters, phosphoamidates,carbamates, etc.), charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), andmodified linkages (e.g., alpha anomeric nucleic acids, etc.). Alsoincluded are synthetic molecules that mimic nucleic acids in theirability to bind to a designated sequence via hydrogen bonding and otherchemical interactions.

“Mutation” is a change in nucleotide sequence within a gene, or outsidethe gene in a regulatory sequence compared to wild type. The change maybe a deletion, substitution, point mutation, mutation of multiplenucleotides, transposition, inversion, frame shift, nonsense mutation orother forms of aberration that differentiate the nucleic acid or proteinsequence from that of a normally expressed gene in a functional cellwhere expression and functionality are within the normally occurringrange.

“Subject” refers to an animal including mammal, including human.

“Wild type FANCD2” refers to a gene that encodes a protein or anexpressed protein capable of being monoubiquinated to form FANCD2-L fromFANCD-S within a cell.

We have found that some Fanconi Anemia has similarities with a group ofsyndromes including ataxia telangiectasia (AT), Xeroderma pigmentosum(XP), Cockayne syndrome (CS), Bloom's syndrome, myelodysplasticsyndrome, aplastic anemia, cancer susceptibility syndromes and HNPCC(see Table 2). These syndromes have an underlying defect in DNA repairand are associated with defects in maintenance of chromosomal integrity.Defects in pathways associated with DNA repair and maintenance ofchromosomal integrity result in genomic instability, and cellularsensitivity to DNA damaging agents such as bifunctional alkylatingagents that cause intrastrand crosslinking. Moreover, deficiencies inDNA repair mechanisms appear to substantially increase the probabilityof initiating a range of cancers through genetic rearrangements. Thisobservation is pertinent with regard to the clinical use of DNAcross-linking drugs including mitomycin C, cisplatin, cyclophosphamide,psoralen and UVA irradiation.

Although Fanconi Anemia is a rare disease, the pleiotropic effects of FAindicate the importance of the wild type function of FA proteins in thepathway for diverse cellular processes including genome stability,apoptosis, cell cycle control and resistance to DNA crosslinks. Thecellular abnormalities in FA include sensitivity to cross-linkingagents, prolongation of G2 phase of cell cycle, sensitivity to oxygenincluding poor growth at ambient O2, overproduction of O2 radicals,deficient O2 radical defense, deficiency in superoxide dismutase;sensitivity to ionizing radiation (G2 specific); overproduction of tumornecrosis factor, direct defects in DNA repair including accumulations ofDNA adducts, and defects in repair of DNA cross-links, genomicinstability including spontaneous chromosome breakage, andhypermutability by deletion mechanism, increased aptosis, defective p53induction, intrinsic stem cell defect, including decreased colony growthin vitro; and decreased gonadal stem cell survival.

These features are reflective of the involvement of FA in maintenance ofhematopoietic and gonadal stem cells, as well as the normal embryonicdevelopment of many different structures, including the skeleton andurogenital systems. Cell samples from patients were analyzed todetermine defects in the FA complementation group D. Lymphoblasts fromone patient gave rise to the PD20 cell line which was found to bemutated in a different gene from HSC62 derived from another patient witha defect in the D complementation group Mutations from both patientsmapped to the D complementation group but to different genes hence thenaming of two FANCD proteins-FANCD1 (HSC62) and FANCD2 (PD20) (Timmerset al., (2001) Molecular Cell, Vol. 7, pp. 241-248). We have shown thatFANCD2 is the endpoint of the FA pathway and is not part of the FAnuclear complex nor required for its assembly or stability and thatFANCD2 exists in two isoforms, FANCD2-S and FANCD2-L. We have also shownthat transformation of the protein short form (FAND2-S) to the proteinlong form (FANCD2-L) occurs in response to the FA complex (FIG. 8).Defects in particular proteins associated with the FA pathway result infailure to make an important post translationally modified form ofFANCD2 identified as FANCD2-L. The two isoforms of FANCD2 are identifiedas the short form and the long form.

Failure to make FANCD2-L correlates with errors in DNA repair and cellcycle abnormalities associated with diseases listed above.

To understand more about the role of FANCD2 in the aforementionedsyndromes, we cloned the FANCD2 gene and determined the proteinsequence. The FANCD2 gene has an open reading frame of 4,353 base pairsand forty four exons which encodes a novel 1451 amino acid nuclearprotein, with a predicted molecular weight of 166 kD. Western blotanalysis revealed the existence of 2 protein isoforms of 162 and 155 kD.The sequence corresponding to the 44 Intron/Exon Junctions are providedin Table 6 (SEQ ID NO:9-94).

Unlike previously cloned FA proteins, FANCD2 proteins from severalnonvertebrate eukaryotes showed highly significant alignment scores withproteins in D. melanogaster, A. thaliana, and C. elegans. The drosophilahomologue, has 28% amino acid identity and 50% similarity to FANCD2(Figure and SEQ ID NO:1-3) and no functional studies have been carriedout in the respective species. No proteins similar to FANCD2 were foundin E. coli or S. cerevisiae.

We obtained the FANCD2 DNA sequence (SEQ ID NO:5) by analyzing thechromosome 3p locus in PD20 and VU008, two FA cell lines havingbiallelic mutations in the FANCD2 gene (FIG. 10). The cell lines wereassigned as complementation group D because lymphoblasts from thepatients failed to complement HSC62, the reference cell line for groupD. FANCD2 mutations were not detected in this group D reference cellline which indicates that the gene mutated in HSC62 is the gene encodingFANCD1 and in PD20 and VU008 is FANCD2 (FIG. 11). Microcell mediatedchromosome transfer was used to identify the mutations (Whitney et al.,Blood, (1995) Vol. 88, 49-58). Detailed analysis of five microcellhybrids containing small overlapping deletions encompassing the locusnarrowed the candidate region of the FANCD2 gene to 200 kb. The FANCD2gene was isolated as follows: Three candidate ESTs were localized in ornear this FANCD2 critical region. Using 5′ and 3′ RACE to obtainfull-length cDNAs, the genes were sequenced, and the expression patternof each was analyzed by northern blot. EST SCC34603 had ubiquitous andlow level expression of a 5 kb and 7 kb mRNA similar to previouslycloned FA genes. Open reading frames were found for TIGR-A004X28,AA609512 and SGC34603 and were 234, 531 and 4413 bp in lengthrespectively. All 3 were analyzed for mutations in PD20 cells bysequencing cloned RT-PCR products. Whereas no sequence changes weredetected in TIGR-A004X28 and AA609512, five sequence changes were foundin SGC34603. Next, we determined the structure of the SGC34603 gene byusing cDNA sequencing primers on BAC 177N7 from the critical region.

Based on the genomic sequence information, PCR primer pairs weredesigned (Table 7), the exons containing putative mutations wereamplified, and allele-specific assays were developed to screen the PD20family as well as 568 control chromosomes. Three of the alleles werecommon polymorphisms; however, 2 changes were not found in the controlsand thus represented potential mutations (Table 3). The first was amaternally inherited A->G change at nt 376. In addition to changing anamino acid (S126G), this alteration was associated with mis-splicing andinsertion of 13 bp from intron 5 into the mRNA. 43/43 (100%)independently cloned RT-PCR products with the maternal mutationcontained this insertion, whereas only 3% ( 1/31) of control cDNA clonesdisplayed mis-spliced mRNA. The 13 bp insertion generated a frame-shiftand predicts a severely truncated protein only 180 aminoacids in length.The second alteration was a paternally inherited missense change atposition 1236 (R1236H). The segregation of the mutations in the PD20core family is depicted in FIG. 10. Because the SGC34603 gene of PD20contained both a maternal and a paternal allele not present on 568control chromosomes and because the maternal mutation was associatedwith mis-splicing in 100% of cDNAs analyzed, we concluded that SGC34603is the FANCD2 gene.

The protein encoded by FANCD2 is absent in PD20: To further confirm theidentity of SGC34603 as FANCD2, an antibody was raised against theprotein, and Western blot analysis was performed (FIG. 11). Thespecificity of the antibody was shown by retroviral transduction andstable expression FANCD2 in PD20 cells (FIG. 11). In wild-type cellsthis antibody detected two bands (155 and 162 kD) which we call FANCD2-Sand -L (best seen in FIG. 11). FANCD2 protein levels were markedlydiminished in all MMC-sensitive cell lines from patient PD20 (FIG. 11 a,lanes 2, 4) but present in all wild-type cell lines and FA cells fromother complementation groups. Furthermore, PD20 cells corrected bymicrocell-mediated transfer of chromosome 3 also made normal amounts ofprotein (FIG. 11 a, lane 3).

Functional complementation of FA-D2 cells with the FANCD2 cDNA: We nextassessed the ability of the cloned FANCD2 cDNA to complement the MMCsensitivity of FA-D2 cells (FIG. 12). The full length FANCD2 cDNA wassubcloned into the retroviral expression vector, pMMP-puro, aspreviously described (Pulsipher et al. (1998), Mol. Med., Vol. 4, pp.468-479). The transduced PD-20 cells expressed both isoforms of theFANCD2 protein, FANCD2-S and FANCD2-L (FIG. 12 c). Transduction of PA-D2(PD20) cells with pMMP-FANCD2 corrected the MMC sensitivity of thecells. These results further show that the cloned FANCD2 cDNA encodesthe FANCD2-S protein, which can be post-translationally-modified to theFANCD2-L isoform. This important modification is discussed in greaterdetail below.

Analysis of a phenotypically reverted PD20 clone: We next generatedadditional evidence demonstrating that the sequence variations in PD20cells were not functionally neutral polymorphisms. Towards this end weperformed a molecular analysis of a revertant lymphoblast clone(PD20-c1.1) from patient PD20 which was no longer sensitive to MMC.Phenotypic reversion and somatic mosaicism are frequent findings in FAand have been associated with intragenic events such as mitoticrecombination or compensatory frame-shifts. Indeed, −60% of maternallyderived SGC34603 cDNAs had a novel splice variant inserting 36 bp ofintron 5 sequence rather than the usually observed 13 bp (FIG. 13). Theappearance of this in-frame splice variant correlated with a de novobase change at position IVS5+6 from G to A (FIG. 13) and restoration ofthe correct reading frame was confirmed by Western blot analysis. Incontrast to all MMC sensitive fibroblasts and lymphoblasts from patientPD20, PD20-c1.1 produced readily detectable amounts of FANCD2 protein ofslightly higher molecular weight than the normal protein.

Analysis of cell lines from other “FANCD” patients: The antibody wasalso used to screen additional FA patient cell lines, including thereference cell line for FA group D, HSC and 2 other cell linesidentified as group D by the European Fanconi Anemia Registry (EUFAR).VU008 did not express the FANCD2 protein and was found to be a compoundheterozygote, with a missense and nonsense mutation, both in exon 12,and not found on 370 control chromosomes (Table 3, FIG. 11). Themissense mutation appears to destabilize the FANCD2 protein, as there isno detectable FANCD2 protein in lysates from VU008 cells. A thirdpatient PD733 also lacked FANCD2 protein (FIG. 11 b, lane 3) and asplice mutation leading to absence of exon 17 and an internal deletionof the protein was found. The correlation of the mutations with theabsence of FANCD2 protein in cell lysates derived from these patientssubstantiates the identity of FANCD2 as a FA gene. In contrast, readilydetectable amounts of both isoforms of the FANCD2 protein were found inHSC62 (FIG. 11 a, lane 8) and VU423 cDNA and genomic DNA from both celllines were extensively analyzed for mutations, and none were found. Inaddition, a whole cell fusion between VU423 and PD20 fibroblasts showedcomplementation of the chromosome breakage phenotype (Table 5). Takentogether these data show that FA group D are genetically heterogeneousand that the gene(s) defective in HSC62 and VU423 are distinct fromFANCD2.

The identification and sequencing of the FANCD2 gene and proteinprovides a novel target for therapeutic development, diagnostic testsand screening assays for diseases associated with failure of DNA repairand cell cycle abnormalities including but not limited to those listedin Table 2.

The following description provides novel and useful insights into thebiological role of FANCD2 in the FA pathway which provides a basis fordiagnosis and treatment of the aforementioned syndromes.

Evidence that FA cells have an underlying molecular defect in cell cycleregulation include the following: (a) FA cells display a cell cycledelay with 4N DNA content which is enhanced by treatment with chemicalcrosslinking agents, (b) the cell cycle arrest and reduced proliferationof FA cells can be partially corrected by overexpression of a protein,SPHAR, a member of the cyclin family of proteins and (c) caffeineabrogates the G2 arrest of FA cells. Consistent with these results,caffeine constitutively activates cdc2 and may override a normal G2 cellcycle checkpoint in FA cells. Finally, the FANCC protein binds to thecyclin dependent kinase, cdc2. We propose that the FA complex may be asubstrate or modulator of the cyclinB/cdc2 complex.

Additionally, evidence that FA cells have an underlying defect in DNArepair is suggested by (a) FA cells that are sensitive to DNAcross-linking agents and ionizing radiation (IR), suggesting a specificdefect in the repair of cross-linked DNA or double strand breaks; (b)DNA damage of FA cells which results in a hyperactive p53 response,suggesting the presence of defective repair yet intact checkpointactivities; and (c) FA cells with a defect in the fidelity ofnon-homologous end joining and an increased rate of homologousrecombination (Garcia-Higuera et al., Mol. Cell., (2001) Vol. 7, pp.249-262), (Grompe et al., Hum. Mol. Genet., (2001) Vol. 10, pp. 1-7).

Despite these general abnormalities in cell cycle and DNA repair, themechanism by which FA pathway regulates these activities has remainedelusive. Here we show that the FANCD2 protein functions downstream ofthe FA protein complex. In the presence of the assembled FA proteincomplex, the FANCD2 protein is activated to a high molecular weight,monoubiquitinated isoform which appears to modulate an S phase specificDNA repair response. The activated FANCD2 protein accumulates in nuclearfoci in response to DNA damaging agents and co-localizes andcoimmunoprecipitates with a known DNA repair protein, BRCA1 Theseresults resolve previous conflicting models of the FA pathway (D'Andreaet al., 1997) and demonstrate that the FA proteins cooperate in acellular response to DNA damage.

The FA pathway includes the formation of the FA multisubunit nuclearcomplex which in addition to A/C/G, we have shown also includes FANCF asa subunit of the complex (FIG. 8). The FA pathway becomes “active”during the S phase to provide S phase specific repair response orcheckpoint response. The normal activation of the FA pathway whichrelies on the FA multisubunit complex results in the regulatedmonoubiquitination of the phosphoprotein-FANCD2 via a phosphorylationstep to a high molecular weight activated isoform identified as FANCD-2L(FIG. 1). Monoubiquitination is associated with cell trafficking.FANCD2-L appears to modulate an S phase specific DNA repair response(FIG. 3). The failure of FA cells to activate the S phase specificactivation of FANCD2 is associated with cell cycle specificabnormalities. The activated FANCD2 protein accumulates in nuclear fociin response to the DNA damaging agents, MMC and IR, and co-localizes andco-immunoprecipitates with a known DNA repair protein, BRCA1 (FIGS.4-6). These results resolve previous conflicting models of FA proteinfunction (D'Andrea et al., 1997) and strongly support a role of the FApathway in DNA repair.

We have identified for the first time, an association between FANCD2isoforms with respect to the FA pathway and proteins that are knowndiagnostic molecules for various cancers. A similar pathway with respectto DNA damage for the BRCA1 protein which is activated to a highmolecular weight, post-translationally-modified isoform in S phase or inresponse to DNA damage suggests that activated FANCD2 protein interactswith BRCA1. More particularly, the regulated monoubiquitination ofFANCD2 appears to target the FANCD2 protein to nuclear foci containingBRCA1. FANCD2 co-immunoprecipitates with BRCA1, and may further bindwith other “dot” proteins, such as RAD50, Mre11, NBS, or RAD51. Recentstudies demonstrate that BRCA1 foci are composed of a large (2Megadalton) multi-protein complex (Wang et al., Genes Dev., (2001) Vol.14, pp. 927-939). This complex includes ATM, ATM substrates involved inDNA repair functions (BRCA1), and ATM substrates involved in checkpointfunctions (NBS). It is further suggested that damage recognition andactivation of the FA pathway involve kinases which respond to DNA damageincluding ATM, ATR, CHK1, or CHK2.

We have found that the DNA damaging reagents, IR and MMC, activateindependent post-translational modifications of FANCD2 result indistinct functional consequences. IR activates the ATM-dependentphosphorylation of FANCD2 at Serine 222 resulting in an S phasecheckpoint response. MMC activates the BRACA-1 dependent and FA pathwaydependent monoubiquitination of FANCD2 at lysine 561, resulting in theassembly of FANCD2/BRCA1 nuclear foci and MMC resistance. FANCD2therefore has two independent functional roles in the maintenance ofchromosomal stability resulting from two discrete post-translationalmodifications provide a link between two additional cancersusceptibility genes (ATM and BRCA1) in a common pathway. Severaladditional lines of evidence support an interaction between FANCD2 andBRCA1. First, the BRCA1 (−/−) cell line, HCC1937 (Scully et al., Mol.Cell, (1999) Vol. 4, pp. 1093-1099) has a “Fanconi Anemia-like”phenotype, with chromosome instability and increased tri-radial andtetra-radial chromosome formations. Second, although FA cells form BRCA1foci (and RAD51 foci) normally in response to IR, BRCA1 (−/−) cells haveno detectable BRCA1 foci and a greatly decreased number of FANCD2 focicompared to normal cells. Functional complementation of BRCA1 (−/−)cells restored BRCA1 foci and FANCD2 foci to normal levels, and restorednormal MMC resistance.

The amount of FANCD2-L is determined in part by the amount of FAND2-Sthat is synthesized from the fancd2 gene and in part by the availabilityof the FA complex to monoubiquinated FANCD2-S to form FANCD2-L. Theassociation of FANCD2-L with nuclear foci including BRCA and ATM anddetermining the role of FANCD2-L in DNA repair make this protein apowerful target for looking at potential cancer development in patientsfor a wide range of cancers. Such cancers include those that arisethrough lesions on chromosome 3p as well as cancers on other chromosomessuch that mutations result in interfering with production of upstreammembers of the FA pathway such as FANCG, FANCC or FANCA. Cancer linesand primary cells from cancer patients including tumor biopsies arebeing screened for FANCD-L and abnormal levels of this protein isexpected to correlate with early diagnosis of disease. Because FANCD2protein is a final step in a pathway to DNA repair, it is envisaged thatany abnormality in a protein in the one or more pathways that lead tothe conversion of FANCD2-S to FANCD-L will be readily detected bymeasuring levels of FANCD2. Moreover, levels of FANCD2 affect how otherproteins such as BRCA and ATM functionally interact in the nucleus withconsequences for the patient. Analysis of levels of FANCD2 in a patientis expected to aid a physician in a clinical decision with respect tounderstanding the class of cancer presented by the patient. Forinstance, if a cancer cell fails to generate the monoubiquinatedFANCD2-L isoform, the cell may have increased chromosome instability andperhaps increased sensitivity to irradiation or chemotherapeutic agents.This information will assist the physician in procedure improvedtreatment for the patient.

Fanconi Anemia is associated not only with a broad spectrum of differentcancers but also with congenital abnormalities. Development of the fetusis a complex but orderly process. Certain proteins have a particularlybroad spectrum of effects because they disrupt this orderly progressionof development. The FA pathway plays a significant role in developmentand disruption of the FA pathway results in a multitude of adverseeffects. Errors in the FA pathway are detectable through the analysis ofthe FAND2-L protein from fetal cells. FANCD2 represents a diagnosticmarker for normal fetal development and a possible target fortherapeutic intervention.

Consistent with the above, we have shown that FANCD2 plays a role in theproduction of viable sperm. FANCD2 forms foci on the unpaired axes ofchromosomes XY bivalents in late pachytene and in diplotene murinespermatocytes (FIG. 7). Interestingly, FANCD2 foci are also seen at theautosomal telomeres in diplonema. Taken together with the knownfertility defects in FA patients and FA-C knockout mice, ourobservations suggest that activated FANCD2 protein is required fornormal progression of spermatocytes through meiosis I. Most of theFANCD2 foci seen on the XY axes were found to co-localize with BRCA1foci, suggesting that the two proteins may function together in meioticcells. Like BRCA1, FANCD2 was detected on the axial (unsynapsed)elements of developing synaptonemal complexes. Since recombinationoccurs in synapsed regions, FANCD2 may function prior to the initiationof recombination, perhaps to help prepare chromosomes for synapsis or toregulate subsequent recombinational events. The relatively synchronousmanner in which FANCD2 assembles on meiotic chromosomes, and forms dotstructures in mitotic cells, suggests a role of FANCD2 in both mitoticand meiotic cell cycle control.

Embodiments of the invention are directed to the use of the posttranslationally modified isoform: FANCD-2L as a diagnostic target fordetermining the integrity of the FA pathway. Ubiquitination of FANCD2and the formation of FANCD2 nuclear foci are downstream events in the FApathway, requiring the function of several FA genes. We have found thatbiallelic mutations of any of the upstream FA genes (FANCA, FANCB,FANCC, FANCE, FANCF and FANCG) block the posttranslational modificationof FANCD2 the unubiquitinated FANCD2 (FANCD2-S) form to theubiquitinated (FANCD2-L). Any of these upstream defects can beoverridden by transfecting cells with FANCD2 cDNA (FIG. 1 a).

We have demonstrated for the first time the existence of FANCD2 and itsrole in the FA pathway. We have shown that FANCD2 accumulates in nuclearfoci in response to DNA damaging agents where it is associated withother DNA repair proteins such as BRCA1 and ATM. We have alsodemonstrated that FANCD2 exists in two isoforms in cells where areduction in one of the two isoforms, FANCD2-L is correlated withFanconi Anemia and with increased cancer susceptibility. We have usedthese findings to propose a number of diagnostic tests for use in theclinic that will assist with patient care.

These tests include: (a) genetic and prenatal counseling for parentsconcerned about inherited Fanconi Anemia in a future offspring or in anexisting pregnancy; (b) genetic counseling and immunodiagnostic testsfor adult humans to determine increased susceptibility to a cancercorrelated with a defective FA pathway; and (c) diagnosing an alreadyexisting cancer in a subject to provide an opportunity for developingtreatment protocols that are maximally effective for the subject whileminimizing side effects.

The diagnostic tests described herein rely on standard protocols knownin the art for which we have provided novel reagents to test for FANCD2proteins and nucleotide sequences. These reagents include antibodiesspecific for FANCD2 isoforms, nucleotide sequences from which vectors,probes and primers have been derived for detecting genetic alterationsin the FANCD2 gene and cells lines and recombinant cells for preservingand testing defects in the FA pathway.

We have prepared monoclonal and polyclonal antibody preparations asdescribed in Example 1 that are specific for FANCD2-L and FANCD2-Sproteins. In addition, FANCD2 isoform specific antibody fragments andsingle chain antibodies may be prepared using standard techniques. Wehave used these antibodies in wet chemistry assays such asimmunoprecipitation assays, for example Western blots, to identifyFANCD2 isoforms in biological samples (FIG. 1). Conventionalimmunoassays including enzyme linked immunosorbent assays (ELISA),radioimmune assays (RIA), immunoradiometric assays (IRMA) andimmunoenzymatic assays (IEMA) and further including sandwich assays mayalso be used. Other immunoassays may utilize a sample of whole cells orlysed cells that are reacted with antibody in solution and optionallyanalyzed in a liquid state within a reservoir. Isoforms of FANCD2 can beidentified in situ in intact cells including cell lines, tissue biopsiesand blood by immunological techniques using for example fluorescentactivated cell sorting, and laser or light microscopy to detectimmunofluorescent cells (FIGS. 1-7, 9-14). For example, biopsies oftissues or cell monolayers, prepared on a slide in a preserved statesuch as embedded in paraffin or as frozen tissue sections can be exposedto antibody for detecting FANCD2-L and then examined by fluorescentmicroscopy.

In an embodiment of the invention, patient-derived cell lines or cancercell lines are analyzed by immunoblotting and immunofluorescence toprovide a novel simple diagnostic test for detecting altered amounts ofFANCD2 isoforms. The diagnostic test also provides a means to screen forupstream defects in the FA pathway and a practical alternative to thecurrently employed DEB/MMC chromosome breakage test for FA, becauseindividuals with upstream defects in the FA pathway are unable toubiquitinate FANCD2. Other assays may be used including assays thatcombine retroviral gene transfer to form transformed patient derivedcell lines (Pulsipher et al., Mol. Med., (1998) Vol. 4, pp. 468-79)together with FANCD2 immunoblotting to provide a rapid subtypinganalysis of newly diagnosed patients with any of the syndromes describedin Table 2, in particular, that of FA.

The above assays may be performed by diagnostic laboratories, or,alternatively, diagnostic kits may be manufactured and sold to healthcare providers or to private individuals for self-diagnosis. The resultsof these tests and interpretive information are useful for thehealthcare provider in diagnosis and treatment of a patient's condition.

Genetic tests can provide for a subject, a rapid reliable risk analysisfor a particular condition against an epidemiological baseline. Our datasuggests that genetic heterogeneity occurs in patients with FA withinthe FANCD2 complementation group. We have found a correlation betweengenetic heterogeneity and disease as well as genetic heterogeneity andabnormal post-translational modifications that result in the presence orabsence of FANCD2-L. This correlation provides the basis for prognostictests as well as diagnostic tests and treatments for any of thesyndromes characterized by abnormal DNA repair. For example, nucleicacid from a cell sample obtained from drawn blood or from other cellsderived from a subject can be analyzed for mutations in the FANCD2 geneand the subject may be diagnosed to have an increased susceptibility tocancer.

We have located the FANCD2 gene at 3p25.3 on chromosome 3p in a regionwhich correlates to a high frequency of cancer. Cytogenetic and loss ofheterozygosity (LOH) studies have demonstrated that deletions ofchromosome 3p occur at a high frequency in all forms of lung cancer(Todd et al., Cancer Res. Vol. 57, pp. 1344-52). For example, homozygousdeletions were found in three squamous cell lines within a region of3p21. Homozygous deletions were also found in a small cell tumor at 3p12and a 3p14.2. (Franklin et al., Cancer Res. (1997), Vol. 57, pp.1344-52). The present mapping of FANCD2 is supportive of the theory thatthis chromosomal region contains important tumor suppressor genes.Further support for this has been provided by a recent publication ofSekine et al., Human Molecular Genetics, (2001) Vol. 10, pp. 1421-1429,who reported localization of a novel susceptibility gene for familialovarian cancer to chromosome 3p22-p25. The reduction or absence ofFANCD2-L is here proposed to be diagnostic for increased risk of tumorsresulting from mutations not only at the FANCD2 site (3p25.3) but alsoat other sites in the chromosomes possibly arising from defects in DNArepair following cell damage arising from exposure to environmentalagents and normal aging processes.

As more individuals and families are screened for genetic defects in theFANCD2 gene, a data base will be developed in which populationfrequencies for different mutations will be gathered and correlationsmade between these mutations and health profile for the individuals sothat the predictive value of genetic analysis will continually improve.An example of an allele specific pedigree analysis for FANCD2 isprovided in FIG. 10 for two families.

Diagnosis of a mutation in the FANCD2 gene may initially be detected bya rapid immunological assay for detecting reduced amounts of FANCD2-Lproteins. Positive samples may then be screened with available probesand primers for defects in any of the genes in the PA pathway. Where adefect in the FANCD2 gene is implicated, primers or probes such asprovided in Table 7 may be used to detect a mutation. In those samples,where a mutation is not detected by such primers or probes, the entireFANCD2 gene may be sequenced to determine the presence and location ofthe mutation in the gene.

Nucleic acid screening assays for use in identifying a genetic defect inthe FANCD2 gene locus may include PCR and non PCR based assays to detectmutations. There are many approaches to analyzing cell genomes for thepresence of mutations in a particular allele. Alteration of a wild-typeFANCD2 allele, whether, for example, by point mutation, deletion orinsertions can be detected using standard methods employing probes (U.S.Pat. No. 6,033,857). Standard methods include: (a) fluorescent in situhybridization (FISH) which may be used on whole intact cells; and (b)allele specific oligonucleotides (ASO) may be used to detect mutationsusing hybridization techniques on isolated nucleic acid (Conner et al.,Hum. Genet., (1989) Vol. 85, pp. 55-74). Other techniques include (a)observing shifts in electrophoretic mobility of single-stranded DNA onnon-denaturing polyacrylamide gels, (b) hybridizing a FANCD2 gene probeto genomic DNA isolated from the tissue sample, (c) hybridizing anallele-specific probe to genomic DNA of the tissue sample, (d)amplifying all or part of the FANCD2 gene from the tissue sample toproduce an amplified sequence and sequencing the amplified sequence, (e)amplifying all or pant of the FANCD2 gene from the tissue sample usingprimers for a specific FANCD2 mutant allele, (f) molecular cloning allor part of the FANCD2 gene from the tissue sample to produce a clonedsequence and sequencing the cloned sequence, (g) identifying a mismatchbetween (i) a FANCD2 gene or a FANCD2 mRNA isolated from the tissuesample, and (ii) a nucleic acid probe complementary to the humanwild-type FANCD2 gene sequence, when molecules (i) and (ii) arehybridized to each other to form a duplex, (h) amplification of FANCD2gene sequences in the tissue sample and hybridization of the amplifiedsequences to nucleic acid probes which comprise wild-type FANCD2 genesequences, (i) amplification of FANCD2 gene sequences in the tissuesample and hybridization of the amplified sequences to nucleic acidprobes which comprise mutant FANCD2 gene sequences, (j) screening for adeletion mutation in the tissue sample, (k) screening for a pointmutation in the tissue sample, (l) screening for an insertion mutationin the tissue sample, and (m) in situ hybridization of the FANCD2 geneof the tissue sample with nucleic acid probes which comprise the FANCD2gene.

It is often desirable to scan a relatively short region of a gene orgenome for point mutations: The large numbers of oligonucleotides neededto examine all potential sites in the sequence can be made by efficientcombinatorial methods (Southern, E. M et al., Nucleic Acids Res., (1994)Vol. 22, pp. 1368-1373). Arrays may be used in conjunction with ligaseor polymerase to look for mutations at all sites in the target sequence(U.S. Pat. No. 6,307,039). Analysis of mutations by hybridization can beperformed for example by means of gels, arrays or dot blots.

The entire gene may be sequenced to identify mutations (U.S. Pat. No.6,033,857). Sequencing of the FANCD2 locus can be achieved usingoligonucleotide tags from a minimally cross hybridizing set which becomeattached to their complements on solid phase supports when attached totarget sequence (U.S. Pat. No. 6,280,935).

Other approaches to detecting mutations in the FANCD2 gene include thosedescribed in U.S. Pat. No. 6,297,010, U.S. Pat. No. 6,287,772 and U.S.Pat. No. 6,300,076. It is further contemplated that the assays mayemploy nucleic acid microchip technology or analysis of multiple samplesusing laboratories on chips. Correlation of these mutations with theresults of genetic studies on breast, ovarian or prostate cancerpatients can then be used to determine if an identified defect withinthe FANC D2 gene is a cancer-associated defect according to theinvention.

A subject who has developed a tumor maybe screened using nucleic aciddiagnostic tests or antibody based tests to detect a FANCD2 genemutation or a deficiency in FANCD2-L protein. On the basis of suchscreening samples may be obtained from subjects having a wide range ofcancers including melanoma, leukemia, astocytoma, glioblastoma,lymphoma, glioma, Hodgkins lymphoma, chronic lymphocyte leukemia andcancer of the pancreas, breast, thyroid, ovary, uterus, testis,pituitary, kidney, stomach, esophagus and rectum. The clinician has animproved ability to select a suitable treatment protocol for maximizingthe treatment benefit for the patient. In particular, the presence of agenetic lesion or a deficiency in FANCD2-L protein may be correlatedwith responsiveness to various existing chemotherapeutic drugs andradiation therapies.

New therapeutic treatments may be developed by screening for moleculesthat modulate the monoubiquitination of FANCD2-S to give rise toFANCD2-L in cell assays (Examples 11-12) and in knock-out mouse models(Example 10). Such molecules may include those that bind directly toFANCD2 or to molecules such as BRACA-2 that appears to interact withBRACA-1 which in turn appears to be activated by FANCD2.

In addition to screening assays that rely on defects in the FANCD2 geneor protein, an observed failure of the ubiquitination reaction that isnecessary for the formation of FANCD2-L may result from a defect in theFA pathway at any point preceding the post translational modification ofFANCD2 including FANCD2-S itself. Knowing the terminal step in thereactions, enables a screening assay to be formulated in which smallmolecules are screened in cells containing “broken FA pathway” or invitro until a molecule is found to repair the broken pathway. Thismolecule can then be utilized as a probe to identify the nature of thedefect. It may further be used as a therapeutic agent to repair thedefect. For example, we have shown that cell cycle arrest and reducedproliferation of FA cells can be partially corrected by overexpressionof a protein, SPHAR, a member of the cyclin family of proteins. This canform the basis of an assay which is suitable as a screen for identifyingtherapeutic small molecules.

Cells which are deficient in the posttranslational modified FANCD2 areparticularly sensitive to DNA damage. These cells may serve as asensitive screen for determining whether a compound (including toxicmolecules) has the capability for damaging DNA. Conversely, these cellsalso serve as a sensitive screen for determining whether a compound canprotect cells against DNA damage.

FA patients and patients suffering from syndromes associated with DNArepair defects die from complications of bone marrow failure. Genetransfer is a therapeutic option to correct the defect. Multiple defectsmay occur throughout the FA pathway. We have shown that the terminalstep is critical to proper functioning of the cell and the organism. Inan embodiment of the invention, correction of defects anywhere in the FApathway may be satisfactorily achieved by gene therapy or by therapeuticagents that target the transformation of FANCD2-S to FANCD2-L so thatthis transformation is successfully achieved.

Gene therapy may be carried out according to generally accepted methods,for example, as described by Friedman in “Therapy for Genetic Disease,”T. Friedman, ed., Oxford University Press (1991), pp. 105-121. Targetedtissues for ex vivo or in vivo gene therapy include bone marrow forexample, hematopoietic stem cells prior to onset of anemia and fetaltissues involved in developmental abnormalities. Gene therapy canprovide wild-type FAND2-L function to cells which carry mutant FANCD2alleles. Supplying such a function should suppress neoplastic growth ofthe recipient cells or ameliorate the symptoms of Fanconi Anemia.

The wild-type FANCD-2 gene or a part of the gene may be introduced intothe cell in a vector such that the gene remains extrachromosomal. Insuch a situation, the gene may be expressed by the cell from theextrachromosomal location. If a gene portion is introduced and expressedin a cell carrying a mutant FANCD-2 allele, the gene portion may encodea part of the FANCD-2 protein which is required for non-neoplasticgrowth of the cell. Alternatively, the wild-type FANCD-2 gene or a partthereof may be introduced into the mutant cell in such a way that itrecombines with the endogenous mutant FANCD-2 gene present in the cell.

Viral vectors are one class of vectors for achieving gene therapy.Viral-mediated gene transfer can be combined with direct in vivo genetransfer using liposome delivery, allowing one to direct the viralvectors to the tumor cells and not into the surrounding nondividingcells. Alternatively, a viral vector producer cell line can be injectedinto tumors (Culver et al., 1992). Injection of producer cells wouldthen provide a continuous source of vector particles. This technique hasbeen approved for use in humans with inoperable brain tumors.

The vector may be injected into the patient, either locally at the siteof the tumor or systemically (in order to reach any tumor cells that mayhave metastasized to other sites). If the transfected gene is notpermanently incorporated into the genome of each of the targeted tumorcells, the treatment may have to be repeated periodically.

Vectors for introduction of genes both for recombination and forextrachromosomal maintenance are known in the art (for example asdisclosed in U.S. Pat. No. 5,252,479 and PCT 93/07282, and U.S. Pat. No.6,303,379) and include viral vectors such as retroviruses, herpesviruses (U.S. Pat. No. 6,287,557) or adenoviruses (U.S. Pat. No.6,281,010) or a plasmid vector containing the FANCD2-L.

A vector carrying the therapeutic gene sequence or the DNA encoding thegene or piece of the gene may be injected into the patient eitherlocally at the site of a tumor or systemically so as to reachmetastasized tumor cells. Targeting may be achieved without furthermanipulation of the vector or the vector may be coupled to a moleculehaving a specificity of binding for a tumor where such molecule may be areceptor agonist or antagonist and may further include a peptide, lipid(including liposomes) or saccharide including an oligopolysaccharide orpolysaccharide) as well as synthetic targeting molecules. The DNA may beconjugated via polylysine to a binding ligand. If the transfected geneis not permanently incorporated into the genome of each of the targetedtumor cells, the treatment may have to be repeated periodically.

Methods for introducing DNA into cells prior to introduction into thepatient may be accomplished using techniques such as electroporation,calcium phosphate coprecipitation and viral transduction as described inthe art (U.S. Pat. No. 6,033,857), and the choice of method is withinthe competence of the routine experimenter.

Cells transformed with the wild-type FANCD2 gene or mutant FANCD2 genecan be used as model systems to study remission of diseases resultingfrom defective DNA repair and drug treatments which promote suchremission.

As generally discussed above, the FANCD2 gene or fragment, whereapplicable, may be employed in gene therapy methods in order to increasethe amount of the expression products of such genes in abnormal cells.Such gene therapy is particularly appropriate for use in pre-cancerouscells, where the level of FANCD2-L polypeptide may be absent ordiminished compared to normal cells and where enhancing the levels ofFANCD2-L may slow the accumulation of defects arising from defective DNArepair and hence postpone initiation of a cancer state. It may also beuseful to increase the level of expression of the FANCD2 gene even inthose cells in which the mutant gene is expressed at a “normal” level,but there is a reduced level of the FANCD2-L isoform. The critical roleof FANCD2-L in normal DNA repair provides an opportunity for developingtherapeutic agents to correct a defect that causes a reduction in levelsof FANCD2-L. One approach to developing novel therapeutic agents isthrough rational drug design. Rational drug design can providestructural analogs of biologically active polypeptides of interest or ofsmall molecules with which they interact (e.g., agonists, antagonists,inhibitors or enhancers) in order to fashion more active or stable formsof the polypeptide, or to design small molecules which enhance orinterfere with the function of a polypeptide in vivo (Hodgson, 1991).Rational drug design may provide small molecules or modifiedpolypeptides which have improved FANCD2-L activity or stability or whichact as enhancers, inhibitors, agonists or antagonists of FANCD2-Lactivity. By virtue of the availability of cloned FANCD2 sequences,sufficient amounts of the FANCD2-L polypeptide may be made available toperform such analytical studies as x-ray crystallography. In addition,the knowledge of the FANCD2-L protein sequence provided herein willguide those employing computer modeling techniques in place of, or inaddition to x-ray crystallography.

Peptides or other molecules which have FANCD2-L activity can be suppliedto cells which are deficient in the protein in a therapeuticformulation. The sequence of the FANCD2-L protein is disclosed forseveral organisms (human, fly and plant) (SEQ ID NO:1-3). FANCD2 couldbe produced by expression of the cDNA sequence in bacteria, for example,using known expression vectors with additional posttranslationalmodifications. Alternatively, FANCD2-L polypeptide can be extracted fromFANCD2-L-producing mammalian cells. In addition, the techniques ofsynthetic chemistry can be employed to synthesize FANCD2-L protein.Other molecules with FANCD2-L activity (for example, peptides, drugs ororganic compounds) may also be used as a therapeutic agent. Modifiedpolypeptides having substantially similar function are also used forpeptide therapy.

Similarly, cells and animals which carry a mutant FANCD2 allele or makeinsufficient levels of FANCD2-L can be used as model systems to studyand test for substances which have potential as therapeutic agents. Thecells which may be either somatic or germline can be isolated fromindividuals with reduced levels of FANCD2-L. Alternatively, the cellline can be engineered to have a reduced levels of FANCD2-L, asdescribed above. After a test substance is applied to the cells, the DNArepair impaired transformed phenotype of the cell is determined.

The efficacy of novel candidate therapeutic molecules can be tested inexperimental animals for efficacy and lack of toxicity. Using standardtechniques, animals can be selected after mutagenesis of whole animalsor after genetic engineering of germline cells or zygotes to formtransgenic animals. Such treatments include insertion of mutant FANCD2alleles, usually from a second animal species, as well as insertion ofdisrupted homologous genes. Alternatively, the endogenous FANCD2 gene ofthe animals may be disrupted by insertion or deletion mutation or othergenetic alterations using conventional techniques (Capecchi, Science,(1989) Vol. 244, pp. 1288-1292) (Valancius and Smithies, 1991). Aftertest substances have been administered to the animals, the growth oftumors must be assessed. If the test substance prevents or suppressespathologies arising from defective DNA repair, then the test substanceis a candidate therapeutic agent for the treatment of the diseasesidentified herein.

The subject invention provides for Fanconi Anemia/BRCA-based diagnosticassays to determine if a patient has cancer or is at an increased riskof cancer. The invention also features screening methods for thediscovery of novel cancer therapeutics that are inhibitors of theFanconi Anemia/BRCA pathway. Finally, the invention provides methods forthe chemosensitization of tumor cells that have become resistant to oneor more chemotherapy compounds as well as assays to determine theefficacy of chemotherapy drugs.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, cell biology,microbiology and recombinant DNA techniques, which are within the skillof the art. Such techniques are explained fully in the literature. See,e.g., Sambrook, Fritsch & Maniatis, 1989, Molecular Cloning: ALaboratory Manual, Second Edition; Oligonucleotide Synthesis (M. J.Gait, ed., 1984); Nucleic Acid Hybridization (B. D. Harnes & S. J.Higgins, eds., 1984); A Practical Guide to Molecular Cloning (B. Perbal,1984); (Harlow, E. and Lane, D.) Using Antibodies: A Laboratory Manual(1999) Cold Spring Harbor Laboratory Press; and a series, Methods inEnzymology (Academic Press, Inc.); Short Protocols In Molecular Biology,(Ausubel et al., ed., 1995). All patents, patent applications, andpublications mentioned herein, both supra and infra, are herebyincorporated by reference in their entirety.

Tissue Biopsies

The invention provides for the preparation of cellular extracts fromtissue biopsies of patients including, but not limited to brain, heart,lung, lymph nodes, eyes, joints, skin and neoplasms associated withthese organs. “Tissue biopsy” also encompasses the collection ofbiological fluids including but not limited to blood, plasma, sputum,urine, cerebrospinal fluid, lavages, and leukophoresis samples. In apreferred embodiment, “tissue biopsies” according to the invention aretaken from tumors of the breast, ovary or prostate. “Tissue biopsies”are obtained using techniques well known in the art including needleaspiration and punch biopsy of the skin.

Cisplatin

Cisplatin has been widely used to treat cancers such as metastatictesticular or ovarian carcinoma, advanced bladder cancer, head or neckcancer, cervical cancer, lung cancer or other tumors. Cisplatin can beused alone or in combination with other agents, with efficacious dosesused in clinical applications of 15-20 mg/m2 for 5 days every threeweeks for a total of three courses. Exemplary doses may be 0.50 mg/m2,1.0 mg/m2, 1.50 mg/m2, 1.75 mg/m2, 2.0 mg/m2, 3.0 mg/m2, 4.0 mg/m2, 5.0mg/m2, 10 mg/m2. Of course, all of these dosages are exemplary, and anydosage in-between these points is also expected to be of use in theinvention. Cisplatin is not absorbed orally and must therefore bedelivered via injection intravenously, subcutaneously, intratumorally orintraperitoneally. Procedures for proper handling and disposal ofanticancer drugs should be considered. Several guidelines on thissubject have been published and are known by those in the art.

For example, PLATINOL-AQ, (cisplatin injection) NDC 0015-3220-22(Bristol Myers Squibb) is supplied as a sterile, multidose vial withoutpreservatives. Each multidose vial contains 50 mg of cisplatin NDC0015-3221-22 and should be stored at 15° C.-25° C. and protected fromlight. The cisplatin remaining in the amber vial following initial entryis stable for 28 days protected from light or for 7 days underfluorescent room light.

The prescribing information for PLATINOL-AQ, (cisplatin injection) NDC0015-3220-22 is available from Bristol Myers Squibb. The plasmaconcentrations of cisplatin decay monoexponentially with a half-life ofabout 20 to 30 minutes following bolus administrations of 50 or 100mg/m2 doses. Monoexponential decay and plasma half-lives of about 0.5hour are also seen following two hour or seven hour infusions of 100mg/m2. After the latter, the total-body clearances and volumes ofdistribution at steady-state for cisplatin are about 15 to 16 L/h/m2 and11 to 12 L/m2.

Dosage and Administration of Cisplatin

The dosage and administration of cisplatin for the treatment of canceris known in the art. The prescribing information of PLATINOL-AQ (BristolMyers Squibb) recommends the following guidelines for dosage andadministration: “Needles or intravenous sets containing aluminum partsthat may come in contact with PLATINOL-AQ should not de used forpreparation or administration. Aluminum reacts with PLATINOL-AQ, causingprecipitate formation and a loss of potency”.

Metastatic Testicular Tumors: The usual PLATINOL-AQ dose for thetreatment of testicular cancer in combination with other approvedchemotherapeutic agents is 20 mg/m2 I.V. daily for 5 days per cycle.

Metastatic Ovarian Tumors: The usual PLATINOL-AQ dose for the treat-mentof metastatic ovarian tumors in combination with CYTOXAN(cy-clophosphamide) is 75-100 mg/m2 I.V. per cycle once every 4 weeks,(Day 1). The dose of CYTOXAN when used in combination with PLATINOL-AQis 600 mg/m2 I.V. once every 4 weeks, (Day 1). For directions for theadministration of CYTOXAN, refer to the CYTOXAN package insert. Incombination therapy, PLATINOL-AQ and CYTOXAN are administeredsequentially. As a single agent, PLATINOL-AQ should be administered at adose of 100 mg/m2 I.V. per cycle once every 4 weeks.

Advanced Bladder Cancer: PLATINOL-AQ (cisplatin injection) should beadministered as a single agent at a dose of 50-70 mg/m2 I.V. per cycleonce every 3 to 4 weeks depending on the extent of prior exposure toradiation therapy and/or prior chemotherapy. For heavily pretreatedpatients an initial dose of 50 mg/m2 per cycle repeated every four weeksis recommended. Pretreatment hydration with 1 to 2 liters of fluidinfused for 8 to 12 hours prior to a PLATINOL-AQ dose is recommended.The drug is then diluted in 2 liters of 5% Dextrose in ½ or ⅓ normalsaline containing 37.5 g of mannitol, and infused over a 6- to 8-hourperiod. If diluted solution is not to be used within 6 hours, protectsolution from light. Do not dilute PLATINOL-AQ in just 5% DextroseInjection. Adequate hydration and urinary output must be maintainedduring the following 24 hours. A repeat course of PLATINOL-AQ should notbe given until the serum creatinine is below 1.5 mg/100 mL, and/or theBUN is below 25 mg/100 mL. A repeat course should not be given untilcirculating blood elements are at an acceptable level(platelets >100,000/mm2, WBC >4,000/mm2). Subsequent doses ofPLATINOL-AQ should not be given until an audiometric analysis indicatesthat auditory acuity is within normal limits. As with other potentiallytoxic compounds, caution should be exercised in handling the aqueoussolution. Skin reactions associated with accidental exposure tocisplatin may occur. The use of gloves is recommended. The aqueoussolution should be used intravenously only and should be administered byI.V. infusion over a 6- to 8-hour period.

Dosage and Administration of a Chemosensitizing Agent

Methods of cancer chemosensitization are reported in U.S. Pat. No.5,776,925, which is incorporated herein in its entirety. Cancertreatment according to the present invention envisions the use of one ormore anti-neoplastic agents in conjunction with compounds that are notnecessarily cytotoxic in themselves, but modify the host or tumor so asto enhance anticancer therapy. Such agents are called chemosensitizers.

Treatment with a chemosensitizing agent is therapeutically effective ina cancer patient, according to the invention, if tumor size is decreasedby 10%, preferably 25%, preferably 50%, more preferably 75%, mostpreferably 100% in the presence of an antineoplastic agent andcorresponding chemosensitizing agent as compared to tumor size aftertreatment with the anti-neoplastic agent but in the absence of thecorresponding chemosenziting agent.

The present invention provides for pharmaceutical compositionscomprising a therapeutically effective amount of a chemosensitizingagent, as disclosed herein, in combination with a pharmaceuticallyacceptable carrier or excipient. The chemosensitizers in accordance withthe invention, may be administered to a patient locally or in anysystemic fashion, whether intravenous, subcutaneous, intramuscular,parenteral, intraperitoneal or oral. Preferably, administration will besystemic in conjunction with or before the administration of one or moreanti-neoplastic agents. In a preferred embodiment, the anti-neoplasticagent is cisplatin that is administered according to protocols wellknown in the art and as described herein.

For oral administration, the chemosensitizing agents useful in theinvention will generally be provided in the form of tablets or capsules,as a powder or granules, or as an aqueous solution or suspension.Tablets for oral use may include the active ingredients mixed withpharmaceutically acceptable excipients such as inert diluents,disintegrating agents, binding agents, lubricating agents, sweeteningagents, flavouring agents, colouring agents and preservatives. Suitableinert diluents include sodium and calcium carbonate, sodium and calciumphosphate, and lactose, while corn starch and alginic acid are suitabledisintegrating agents. Binding agents may include starch and gelatin,while the lubricating agent, if present, will generally be magnesiumstearate, stearic acid or talc. If desired, the tablets may be coatedwith a material such as glyceryl monostearate or glyceryl distearate, todelay absorption in the gastrointestinal tract.

Capsules for oral use include hard gelatin capsules in which the activeingredient is mixed with a solid diluent, and soft gelatin capsuleswherein the active ingredients is mixed with water or an oil such aspeanut oil, liquid paraffin or olive oil.

For subcutaneous and intravenous use, the chemosensitizing agents of theinvention will generally be provided in sterile aqueous solutions orsuspensions, buffered to an appropriate pH and isotonicity. Suitableaqueous vehicles include Ringer's solution and isotonic sodium chloride.Aqueous suspensions according to the invention may include suspendingagents such as cellulose derivatives, sodium alginate,polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such aslecithin. Suitable preservatives for aqueous suspensions include ethyland n-propyl p-hydroxybenzoate.

The chemosensitizing agents useful according to the invention may alsobe presented as liposome formulations.

In general a suitable dose will be in the range of 0.01 to 100 mg perkilogram body weight of the recipient per day, preferably in the rangeof 0.2 to 10 mg per kilogram body weight per day. The desired dose ispreferably presented once daily, but may be dosed as two, three, four,five, six or more sub-doses administered at appropriate intervalsthroughout the day. These sub-doses may be administered in unit dosageforms, for example, containing 10 to 1500 mg, preferably 20 to 1000 mg,and most preferably 50 to 700 mg of active ingredient per unit dosageform. Dosages of chemosensitizing agents useful according to theinvention will vary depending upon the condition to be treated orprevented and on the identity of the chemosensitizing agent being used.Estimates of effective dosages and in vivo half-lives for the individualcompounds encompassed by the invention can be made on the basis of invivo testing using an animal model, such as the mouse model describedherein or an adaptation of such method to larger mammals.

In addition to their administration singly, the compounds usefulaccording to the invention can be administered in combination with otherknown chemosensitizing agents and anti-neoplastic agents, as describedherein. In any event, the administering physician can adjust the amountand timing of drug administration on the basis of results observed usingstandard measures of cancer activity known in the art.

Anti-Neoplastic Agents

Nonlimiting examples of anti-neoplastic agents include, e.g.,antimicrotubule agents, topoisomerase inhibitors, antimetabolites,mitotic inhibitors, alkylating agents, intercalating agents, agentscapable of interfering with a signal transduction pathway, agents thatpromote apoptosis, radiation, and antibodies against othertumor-associated antigens (including naked antibodies, immunotoxins andradioconjugates). Examples of the particular classes of anti-canceragents are provided in detail as follows: antitubulin/antimicrotubule,e.g., paclitaxel, vincristine, vinblastine, vindesine, vinorelbin,taxotere; topoisomerase I inhibitors, e.g., topotecan, camptothecin,doxorubicin, etoposide, mitoxantrone, daunorubicin, idarubicin,teniposide, amsacrine, epirubicin, merbarone, piroxantronehydrochloride; antimetabolites, e.g., 5-fluorouracil (5-FU),methotrexate, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate,cytarabine/Ara-C, trimetrexate, gemcitabine, acivicin, alanosine,pyrazofurin, N-Phosphoracetyl-L-Asparate, i.e., PALA, pentostatin,5-azacitidine, 5-Aza 2′-deoxycytidine, ara-A, cladribine,5-fluorouridine, FUDR, tiazofurin,N-[5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl]-L-glutamicacid; alkylating agents, e.g., cisplatin, carboplatin, mitomycin C,BCNU, i.e., Carmustine, melphalan, thiotepa, busulfan, chlorambucil,plicamycin, dacarbazine, ifosfamide phosphate, cyclophosphamide,nitrogen mustard, uracil mustard, pipobroman, 4-ipomeanol; agents actingvia other mechanisms of action, e.g., dihydrolenperone, spiromustine,and desipeptide; biological response modifiers, e.g., to enhanceanti-tumor responses, such as interferon; apoptotic agents, such asactinomycin D; and anti-hormones, for example anti-estrogens such astamoxifen or, for example antiandrogens such as4′-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3′-(trifluoromethyl)propionanilide.

An anti-neoplastic agent is therapeutic in a cancer patient, accordingto the invention, if tumor size is decreased by 10%, preferably 25%,preferably 50%, more preferably 75%, most preferably 100% when comparedto tumor size prior to the initiation of treatment with ananti-neoplastic agent.

In a further embodiment, an anti-neoplastic agent, according to theinvention, is therapeutically effective if the cancer patient remainscancer free, i.e., without any detectable tumors, for preferably 6months, preferably 1 year, more preferably 2 years and most preferably 5years or more after initiation of cancer therapy.

Inhibitors of the Fanconi Anemia/BRCA Pathway According to the Invention

Potential inhibitors of the Fanconi Anemia/BRCA pathway include, but arenot limited to, biomolecules that disrupt the expression or function ofFanconi Anemia/BRCA pathway genes or proteins as defined herein.Potential inhibitors of the Fanconi Anemia/BRCA pathway include, but arenot limited, to Fanconi Anemia/BRCA pathway gene antisense nucleic acids(antisense Fanconi Anemia/BRCA pathway gene RNAs, oligonucleotides,modified oligonucleotides, RNAi), dominant negative mutants of theFanconi Anemia/BRCA pathway gene pathway as well as inhibitors ofFanconi Anemia/BRCA pathway gene transcription, mRNA processing, mRNAtransport, protein translation, protein modification, protein transport,nuclear transport and Fanconi Anemia/BRCA protein complex formation.

In a most preferred embodiment, the present invention provides for smallmolecule inhibitors of the FANC-D2 ubiquitin E3 ligase.

Microarrays According to the Invention

To identify cancer therapeutics or chemosensitizing agents, theinvention provides for the use of microarrays.

In one embodiment, the microarray of the invention is used to identifychemosensitizing agents.

In another embodiment, the microarray of the invention is used to testtissue biopsy samples for the presence of cancer-associated defectswithin the Fanconi Anemia/BRCA pathway genes.

In another embodiment, the microarrays of the invention are used toscreen for inhibitors of the Fanconi Anemia/BRCA gene pathway.

In another embodiment, the microarrays of the invention are to be usedto screen for inhibitors of the FANC-D2 ubiquitin E3 ligase.

In another embodiment, the invention provides for tissue microarrayscomprising tissue biopsy samples from patients who have a cancer or whomay be at risk of cancer that are screening for the presence of cancerassociated defects within Fanconi Anemia/BRCA gene pathway as definedherein. In a preferred embodiment, the tissue microarrays of the presentinvention are used to screen for the presence of mon-ubiqutinated FANCD2-L.

In another embodiment, the invention provides for tissue microarrayscomprising tissue biopsy samples from patients having BRCA-1 andBRCA-2/FANC D-1 cancer-associated defects.

In another embodiment, the invention provides for tissue microarrayscomprising tissue biopsy samples from patients that do not have BRCA-1and BRCA-2/FANC D-1 cancer-associated defects.

A “sequencing array” contains regions of the entire open reading frameof the genes in question, in order to look for mutations in the clincialsample. A “transcriptional profiling array” can have sequences from the3′ end of the genes in questions, in order to determine the expressionof mRNAs in the clinical sample.

A transcriptional profiling array will be used to look at mRNA levelscorresponding to each of the genes in the pathway. For instance, abreast or ovarian cancer which has a decrease in one of the transcripts,e.g., corresponding to FANC F would show that there is a defect in theFanconi Anemia/BRCA pathway, due to decreased FANCF expression.

Construction of a Microarray Substrate of the Microarray

In one embodiment of the invention, the microarray or array comprises asubstrate to facilitate handling of the microarray through a variety ofmolecular procedures. As used herein, “molecular procedure” refers tocontact of the microarray with a test reagent or molecular probe such asan antibody, nucleic acid probe, enzyme, chromagen, label, and the like.In one embodiment, a molecular procedure comprises a plurality ofhybridizations, incubations, fixation steps, changes of temperature(from −4° C. to 100° C.), exposures to solvents, and/or wash steps.

In a further embodiment of the invention, the microarray comprises asubstrate to facilitate exposure of tissue biopsy samples to differentpotential inhibitors of the Fanconi Anemia/BRCA pathway, cancertherapeutics or chemosensitizing agents.

In one embodiment of the invention, the microarray substrate is solventresistant. In another embodiment of the invention, the substrate istransparent. The substrate may be biological, non-biological, organic,inorganic, or a combination of any of these, existing as particles,strands, precipitates, gels, sheets, tubing, spheres, beads, containers,capillaries, pads, slices, films, plates, slides, chips, etc. Thesubstrate is preferably flat or planar but may take on a variety ofalternative surface configurations. The substrate may be a polymerizedLangmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO2,SIN4, modified silicon, or other nonporous substrate, plastic, such aspolyolefin, polyamide, polyacarylamide, polyester, polyacrylic ester,polycarbonate, polytetrafluoroethylene, polyvinyl acetate, and a plasticcomposition containing fillers (such as glass fillers), extenders,stabilizers, and/or antioxidants; celluloid, cellophane or ureaformaldehyde resins or other synthetic resins such as cellulose acetateethylcellulose, or other transparent polymer. Other substrate materialswill be readily apparent to those of skill in the art upon review ofthis disclosure.

In one embodiment, the microarray substrate is rigid; however, inanother embodiment, the profile array substrate is semi-rigid orflexible (e.g., a flexible plastic comprising polycarbonate, cellularacetate, polyvinyl chloride, and the like). In a further embodiment, thearray substrate is optically opaque and substantially non-fluorescent.Nylon or nitrocellulose membranes can also be used as array substratesand include materials such as polycarbonate, polyvinylidene fluoride(PVDF), polysulfone, mixed esters of cellulose and nitrocellulose, andthe like.

The size and shape of the substrate may generally be varied. Thesubstrate may have any convenient shape, such as a disc, square, sphere,circle, etc. However, preferably, the substrate fits entirely on thestage of a microscope. In one embodiment, the profile array substrate isplanar. In one embodiment of the invention, the microarray substrate is1 inch by 3 inches, 77×50 mm, or 22×50 mm. In another embodiment of theinvention, the microarray substrate is at least 10-200 mm×10-200 mm.

Additional Features of the Substrate

In one embodiment of the invention, the substrate comprises a locationfor placing an identifier (e.g., a wax pencil or crayon mark, an etchedmark, a label, a bar code, a microchip for transmitting radio orelectronic signals, and the like). In one embodiment, the locationcomprises frosted glass. In one embodiment, the microchip communicateswith a processor which comprises or can access stored informationrelating to the identity and address of sublocations on the array,and/or including information regarding the individual from whom thetissue was taken, e.g., prognosis, diagnosis, medical history, familymedical history, drug treatment, age of death and cause of death, andthe like.

Sublocations

The microarray comprises a plurality of sublocations. Each sublocationcomprises a tissue stably associated therewith (e.g., able to retain itsposition relative to another sublocation after exposure to at least onemolecular procedure). In one embodiment, the tissue is a tissue whichhas morphological features substantially intact which can be at leastviewed under a microscope to distinguish subcellular features (e.g.,such as a nucleus, an intact cell membrane, organells, and/or othercytological features), i.e., the tissue is not lysed.

In one embodiment of the invention, the microarray comprises from 2-1000sublocations. In another embodiment, the microarray comprises 2, 5, 10,20, 25, 30, 45, 50, 55, 60, 65, 75, 100, 150, 200, 250, 500, 550, 600,650, 700, 750, 800, 850, 900, 950 or 1000 or more sublocations. In oneembodiment of the invention, each sublocation is from 2-10 mm apart. Inanother embodiment of the invention, each sublocation comprises at leastone dimension which is 20-600 mm. The sublocations can be organized inany pattern, and each sublocation can be generally any shape (square,circular, oval, elliptical, disc shaped, rectangular, triangular, andthe like).

In a preferred embodiment, the sublocations are positioned in a regularrepeating pattern (e.g., rows and columns) such that the identificationof each sublocation as to tissue type can be ascertained by the use ofan array locator. In one embodiment, the array locator is a templatehaving a plurality of shapes, each shape corresponding to the shape ofeach sublocation in the array, and maintaining the same relationships aseach sublocation on the array. The array locator is marked bycoordinates, allowing the user to readily identify a sublocation on thearray by virtue of unique coordinates. In one embodiment of theinvention, the array locator is a transparent sheet (e.g., plastic,acetate, and the like). In another embodiment of the invention, thearray locator is a sheet comprising a plurality of holes, each holecorresponding in shape and location to each sublocation on the array.

In one aspect, the invention provides for arrays wherein the compoundscomprising the array are spotted onto a solid support, e.g., spottedusing a robotic GMS 417 arrayer (Affymetrix, Calif.). Alternatively,spotting may be carried out using contact printing technology or othermethods known in the art.

Types of Microarrays According to the Invention Small Molecule Arrays

In the small molecule microarrays or arrays of the invention, the smallmolecules are stably associated with the surface of a solid support,wherein the support may be a flexible or rigid solid support. By “stablyassociated” is meant that each small molecule maintains a uniqueposition relative to the solid support under binding and washingconditions. As such, the samples are non-covalently or covalently stablyassociated with the support surface. Examples of non-covalentassociation include non-specific adsorption, binding based onelectrostatic interactions (e.g., ion pair interactions), hydrophobicinteractions, hydrogen bonding interactions, specific binding through aspecific binding pair member covalently attached to the support surface,and the like. Examples of covalent binding include covalent bonds formedbetween the small molecules and a functional group present on thesurface of the rigid support (e.g., —OH), where the functional group maybe naturally occurring. The surface of the substrate can be preferablyprovided with a layer of linker molecules, although it will beunderstood that the linker molecules are not required elements of theinvention. The linker molecules are preferably of sufficient length topermit small molecules of the invention and on a substrate to bind tosmall molecules and to interact freely with molecules exposed to thesubstrate.

The amount of small molecule present in each composition will besufficient to provide for adequate binding and detection of target smallmolecules during the assay in which the array is employed. Generally,the amount of each small molecule stably associated with the solidsupport of the array is at least about 0.1 pg, preferably at least about0.5 pg and more preferably at least about 1 pg, where the amount may beas high as 1000 pg or higher, but will usually not exceed about 100 pg.In a preferred embodiment, the microarray has a density exceeding 1, 2,5, 7, 10, 15 or 20 or more small molecules/cm2.

Tissue Microarrays

In a preferred embodiment of the invention, the microarrays or arrayscomprise human tissue samples. The microarrays according to theinvention comprise a plurality of sublocations, each sublocationcomprising a tissue sample having at least one known biologicalcharacteristic (e.g., such as tissue type). In a preferred embodiment ofthe invention, the plurality of sublocations comprise cancerous tissueat different neoplastic stages.

In one embodiment of the invention, the cancerous cells at individualsublocations are from an individual with an underlying cancer orpredisposition to having a cancer.

In one embodiment of the invention, the cancerous cells at individualsublocations are from an individual with cancer-associated defects inthe BRCA-1 and/or FANC D1/BRCA-2 genes.

In one embodiment, the microarray comprises at least one sublocationcomprising cancerous cells from a single patient and comprises aplurality of sublocations comprising cells from other tissues and organsfrom the same patient. In a different embodiment, a microarray isprovided comprising cells from a plurality of individuals who have alldied from the same pathology, or from individuals being treated with thesame drug (including those who recovered from the disease and/or thosewho did not).

In another embodiment of the invention, the microarray comprises aplurality of sublocations comprising cells from individuals sharing atrait in addition to cancer. In one embodiment of the invention, thetrait shared is gender, age, a pathology, predisposition to a pathology,exposure to an infectious disease (e.g., HIV), kinship, death from thesame illness, treatment with the same drug, exposure to chemotherapy orradiotherapy, exposure to hormone therapy, exposure to surgery, exposureto the same environmental condition, the same genetic alteration orgroup of alterations, expression of the same gene or sets of genes.

In a further embodiment of the invention, each sublocation of themicroarray comprises cells from different members of a pedigree sharinga family history of cancer (e.g., selected from the group consisting ofsibs, twins, cousins, mothers, fathers, grandmothers, grandfathers,uncles, aunts, and the like). In another embodiment of the invention,the “pedigree microarray” comprises environment-matched controls (e.g.,husbands, wives, adopted children, stepparents, and the like). In stilla further embodiment of the invention, the microarray is a reflection ofa plurality of traits representing a particular patient demographicgroup of interest, e.g., overweight smokers, diabetics with peripheralvascular disease, individuals having a particular predisposition todisease (e.g., sickle cell Anemia, Tay Sachs, severe combinedimmunodeficiency), wherein individuals in each group have cancer.

In a preferred embodiment of the invention, the microarrays comprisehuman tissue biopsies.

FANC D2−/− as disclosed herein. In one embodiment, the microarraycomprises multiple tissues from such a mouse. In another embodiment ofthe invention, the microarray comprises tissues from mice that are FANCD2−/− as disclosed herein, and which have been treated with a cancertherapy (e.g., drugs, antibodies, protein therapies, gene therapies,antisense therapies, and the like).

Screening of Chemosensitizing Agents and Novel Cancer Therapeutics

The microarrays of the invention are used to screen for chemosensitizingagents and cancer therapeutics. The screening procedures used aredisclosed in Examples 15 and 16.

Measurement of Resistance to a Chemotherapy Agent

Methylation of the FANC F gene within tumor cells that are treated withcisplatin results in the repression of FANC F gene expression andthereby causes a disruption in the tumor cell's DNA damage repairmechanisms and resulting in resistance to cisplatin. The inventiontherefore provides for the determination of the methylation state of anyof the Fanconi Anemia/BRCA pathway genes (see Example 19). In apreferred embodiment, the invention provides microarrays of tissuebiopsy samples from patients being treated with one or more chemotherapycompounds for the determination of the methylation state of the FanconiAnemia/BRCA genes as a measurement of the degree of a tumor's resistanceto one or more chemotherapy compounds. Methods of measuring DNAmethylation of genes are well known in the art (see U.S. Pat. Nos.6,200,756; 6,331,393; 6,251,594).

Kits According to the Invention

The invention provides for kits useful for screening forchemosensitizers and cancer therapeutics, as well as kits useful fordiagnosis of cancer or predisposition toward cancer involvingcancer-associated defects in the Fanconi Anemia/BRCA gene pathway. Kitsuseful according to the invention include isolated FANC D2polynucleotide primer pairs, probes, inhibitors of the FanconiAnemia/BRCA pathway and a FANC D2-specific antibody. In addition, kitscan contain control unmethylated FANC D2 genes. In a further embodiment,a kit according to the invention can contain an ovary cancer tumor cellline. All kits according to the invention will comprise the stated itemsor combinations of items and packaging materials therefore. Kits willalso include instructions for use.

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.

EXAMPLES Example 1 Experimental Protocols Used in Examples 2-8

Cell Lines and Culture Conditions. Epstein-Barr virus (EBV) transformedlymphoblasts were maintained in RPMI media supplemented with 15%heat-inactivated fetal calf serum (FCS) and grown in a humidified 5%CO2-containing atmosphere at 37° C. A control lymphoblast line (PD7) andFA lymphoblast lines (FA-A (HSC72), FA-C(PD-4), FA-D (PD-20), FA-F(EUFA121), and FA-G (EUFA316)) have been previously described (de Winteret al., Nat. Genet., (1998) Vol. 20, pp. 281-283) (Whitney et al., Nat.Genet., (1995) Vol. 11, pp. 341-343) (Yamashita et al., P.N.A.S., (1994)Vol. 91, pp. 6712-6716) (de Winter et al., Am. J. Hum. Genet., (2001),Vol. 57, pp. 1306-1308). PD81 is a lymphoblast cell line from an FA-Apatient. The SV40-transformed FA fibroblasts, GM6914, PD426, FAG326SVand PD20F, as well as HeLa cells, were grown in DMEM supplemented with15% FCS. FA cells (both lymphoblasts and fibroblasts) were functionallycomplemented with pMMP retroviral vectors containing the correspondingFANC cDNAs, and functional complementation was confirmed by the MMCassay (Garcia-Higuera et al., Mol. Cell. Biol., (1999) Vol. 19, pp.4866-4873) (Kuang et al., Blood, (2000), Vol. 96, pp. 1625-1632).

Cell Cycle Synchronization. HeLa cells, GM6914 cells, and GM6914 cellscorrected with the pMMP-FANCA retrovirus were synchronized by the doublethymidine block method as previously described, with minor modifications(Kupfer et al., Blood, (1997) Vol. 90, pp. 1047-1054). Briefly, cellswere treated with 2 mM thymidine for 18 hours, thymidine-free media for10 hours, and additional 2 mM thymidine for 18 hours to arrest the cellcycle at the G1/S boundary. Cells were washed twice with PBS and thenreleased in DMEM+15% FCS and analyzed at various time intervals.

Alternatively, HeLa cells were treated with 0.5 mM mimosine (Sigma) for24 hours for synchronization in late G1 phase (Krude, 1999), washedtwice with PBS, and released into DMEM+15% FCS. For synchronization in Mphase, a nocodazole block was used (Ruffner et al., Mol. Cell. Biol.,(1999) Vol. 19, pp. 4843-4854). Cells were treated with 0.1 μg/mlnocodazole (Sigma) for 15 hours, and the non-adherent cells were washedtwice with PBS and replated in DMEM+15%.

Cell Cycle Analysis. Trypsinized cells were resuspended in 0.5 ml of PBSand fixed by adding 5 ml of ice-cold ethanol. Cells were next washedtwice with PBS with 1% bovine serum albumin fractionV (1% BSA/PBS)(Sigma), and resuspended in 0.24 ml of 1% BSA/PBS. After adding 30 μl of500 g/ml propidium iodide (Sigma) in 38 mM sodium citrate (pH7.0) and 30μl of 10 mg/ml DNase free RNaseA (Sigma), samples were incubated at 37°C. for 30 min. DNA content was measured by FACScan (Beckton Dickinson),and data were analyzed by the CellQuest and Modfit LT program (BectonDickinson).

Generation of an anti-FANCD2 antiserum. A rabbit polyclonal antiserumagainst FANCD2 was generated using a GST-FANCD2 (N-terminal) fusionprotein as an antigen source. A 5′ fragment was amplified by polymerasechain reaction (PCR) from the full length FANCD2 cDNA with the primers(SEQ ID NO:95) DF4EcoRI (5′ AGCCTCgaattcGTTTCCAA AAGAAGACTGTCA-3′) and(SEQ ID NO:96) DR816Xh (5′-GGTATCctcgagTCAAGACGA CAACTTATCCATCA-3′). Theresulting PCR product of 841 bp, encoding the amino-terminal 272 aminoacids of the FANCD2 polypeptide was digested with EcoRI/XhoI andsubcloned into the EcoRI/XhoI sites of the plasmid pGEX4T-1 (Pharmacia).A GST-FANCD2 (N-terminal) fusion protein of the expected size (54 kD)was expressed in E. coli strain DH5γ, purified overglutathione-S-sepharose, and used to immunize a New Zealand Whiterabbit. An FANCD2-specific immune antiserum was affinity-purified bypassage over an AminoLink Plus column (Pierce) loaded with GST proteinand by passage over an AminoLink Plus column loaded with the GST-FANCD2(N-terminal) fusion protein.

Generation of anti-FANCD2 MoAbs. Two anti-FANCD2 monoclonal antibodieswere generated as follows. Balb/c mice were immunized with a GST-FANCD2(N-terminal) fusion protein, which was the same fusion protein used forthe generation of the rabbit polyclonal antiserum (E35) against FANCD2.Animals were boosted with immunogen for the four days before fusion,splenocytes were harvested, and hybridization with myeloma cells wasperformed. Hybridoma supernatants were collected and assayed usingstandard ELISA assay as the initial screen and immunoblot analysis ofFANCD2 as the secondary screen.

Two anti-human FANCD2 monoclonal antibodies (MoAbs) (FI17 and FI14) wereselected for further study. Hybridoma supernatants from the two positivecell lines were clarified by centrifugation. Supernatants were used asMoAbs for western blotting. MoAbs were purified using an affinity columnfor IgG. MoAbs were stored as 0.5 mg/ml stocks in phosphate bufferedsaline (PBS). Anti-HA antibody (HA.11) was from Babco.

Immunoblotting. Cells were lysed with 1× sample buffer (50 mM Tris-HClpH6.8, 86 mM 2-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), boiledfor 5 min, and subjected to 7.5% polyacrylamide SDS gel electrophoresis.After electrophoresis, proteins were transferred to nitrocellulose usinga submerged transfer apparatus (BioRad) filled with 25 mM Tris base, 200mM glycine, 20% methanol. After blocking with 5% non-fat dried milk inTBS-T (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween 20) the membranewas incubated with the primary antibody diluted in TBS-T (1:1000dilution for the affinity-purified anti-FANCD2 polyclonal antibody (E35)or anti-HA (HA.11), 1:200 dilution for the anti-FANCD2 mouse monoclonalantibody FI17), washed extensively and incubated with the appropriatehorseradish peroxidase-linked secondary antibody (Amersham).Chemiluminescence was used for detection.

Generation of DNA Damage. Gamma irradiation was delivered using a Gammacell 40 apparatus. UV exposure was achieved using a Stratalinker(Stratagene) after gently aspirating the culture medium. For Mitomycin Ctreatment cells were continuously exposed to the drug for the indicatedtime. Hydroxyurea (Sigma) was added to a final concentration of 1 mM for24 hours.

Detection of Monoubiquitinated FANCD2. HeLa cells (or the FA-Gfibroblasts, FAG326SV) were transfected using FuGENE6 (Roche), followingthe manufacturer's protocol. HeLa cells were plated onto 15 cm tissueculture dishes and were transfected with 15 μg of a HA-tagged ubiquitinexpression vector (pMT 123) (Treier et al., Cell, (1994) Vol. 78, pp.787-798) per dish. Twelve hours following transfection, cells weretreated with the indicated concentration of MMC (0, 10, 40, 160 ng/ml)or the indicated dose of IR (0, 5, 10, 10, 20 Gy). After 24hour-incubation with MMC, or two hours after IR treatment, whole cellextracts were prepared in Lysis Buffer (50 mM TrisHCl pH 7.4, 150 mMNaCl, 1% (v/v) Triton X-100) supplemented with protease inhibitors (1μg/ml leupeptin and pepstatin, 2 μg/ml aprotinin, 1 mMphenylmethylsulfonylfluoride) and phosphatase inhibitors (1 mM sodiumorthovanadate, 10 mM sodium fluoride). Using the polyclonal antibody toFANCD2 (E35), immunoprecipitation (IP) was performed essentially asdescribed (Kupfer et al., 1997) except that each IP was normalized tocontain 4 mg of protein. As a negative control, preimmune serum from thesame rabbit was used in IP reaction. Immunoblotting was done usinganti-HA (HA.11), or anti-FANCD2 (FI17) monoclonal antibody.

Ubiquitin Aldehyde Treatment. HeLa cells were treated with 1 mMhydroxyurea for 24 hours, and whole cell extracts were prepared in LysisBuffer supplemented with protease inhibitors and phosphatase inhibitors.200 pg of cell lysate in 67 μl of reaction with 6.7 μl of 25 μMubiquitin aldehyde (BostonBiochem) in DMSO or with 6.7 μl of DMSO wereincubated at 30° C. or at 37° C. for the indicated periods. Sixty-sevenmicroliters of 2× sample buffer was added to each sample, and thesamples were boiled for 5 min, separated by 7.5% SDS-PAGE, andimmunoblotted for FANCD2 using the FI17 monoclonal anti-human FANCD2antibody.

Immunofluorescence Microscopy. Cells were fixed with 2% paraformaldehydein PBS for 20 min, followed by permeabilization with 0.3% Triton-X-100in PBS (10 min). After blocking in 10% goat serum, 0.1% NP-40 in PBS(blocking buffer), specific antibodies were added at the appropriatedilution in blocking buffer and incubated for 2-4 hours at roomtemperature. FANCD2 was detected using the affinity-purified E35polyclonal antibody (1/100). For BRCA1 detection, we used a commercialmonoclonal antibody (D-9, Santa Cruz) at 2 μg/ml. Cells weresubsequently washed three times in PBS+0.1% NP-40 (10-15 min each wash)and species-specific fluorescein or Texas red-conjugated secondaryantibodies (Jackson Immunoresearch) were diluted in blocking buffer(anti-mouse 1/200, anti-rabbit 1/1000) and added. After 1 hour at roomtemperature three more 10-15 min washes were applied and the slides weremounted in Vectashield (Vector laboratories). Images were captured on aNikon microscope and processed using Adobe Photoshop software.

Meiotic Chromosome Staining. Surface spreads of pachytene and diplotenespermatocytes from male mice between the ages of 16 and 28 days old wereprepared as described by (Peters et al., 1997). A polyclonal goatantibody to the mouse SCP3 protein was used to visualize axial elementsand synaptonemal complexes in the meiotic preparations. The M118 mousemonoclonal antibody against mouse BRCA1 was generated by standardtechniques, by immunizing mice with murine BRCA1 protein. Theaffinity-purified E35 rabbit polyclonal antibody was used in 1:200dilution to detect FANCD. Antibody incubation and detection procedureswere a modification of the protocol of (Moens et al., J. Cell. Biol.,(1987) Vol. 105, pp. 93-103) as described by (Keegan et al., Genes Dev.,(1996) Vol. 10, pp. 2423-2437). Combinations of donkey-anti mouseIgG-FITC-congugated, Donkey-anti rabbit IgG-TRITC-congugated, andDonkey-anti goat IgGCy5-congugated secondary antibodies were used fordetection (Jackson ImmunoResearch Laboratories). All preparations werecounterstained with 4′,6′ diamino-2-phenylindole (DAPI, Sigma) andmounted in a DABCO (Sigma) antifade solution. The preparations wereexamined on a Nikon E1000 microscope (60×CFI Plan Apochromat and 100× CRPlan Fluor oil-immersion objectives). Each fluorochrome (FITC, TRITC,Cy5 and DAPI) image was captured separately as an 800×1000 pixel 12-bitsource image via IPLab software (Scanalytics) controlling a cooled-CCDcamera (Princeton Instruments MicroMax) and the separate 12 bit greyscale images were resampled, 24-bit pseudocolored and merged using AdobePhotoshop.

Example 2 The FA Genes Interact in a Common Cellular Pathway

Normal lymphoblasts express two isoforms of the FANCD2 protein, a shortform (FANCD2-S, 155 kD) and a long form (FANCD2-L, 162 kD). FIG. 1 showswhat happened when whole cell extracts were prepared from a lymphoblastline and cellular proteins were immunoprecipitated with an anti-FANCD2antiserum. Normal wild type cells expressed two isoforms of the FANCD2protein—a low molecular weight isoform FANCD2-S (155 kD isoform) and ahigh molecular weight isoform (FANCD2-L) (162 kD isoform). FANCD2-S isthe primary translation product of the cloned FANCD2 cDNA. We nextevaluated a large series of FA lymphoblasts and fibroblasts forexpression of the FANCD2 isoforms (Table 5). Correction of these FA celllines with the corresponding FA cDNA resulted in functionalcomplementation and restoration of the high molecular weight isoform,FANCD2-L.

As previously described, FA cells are sensitive to the DNA crosslinkingagent, MMC, and in some cases, to ionizing radiation (IR).Interestingly, FA cells from multiple complementation groups (A, C, G,and F) only expressed the FANCD2-S isoform (FIG. 1A, lanes 3, 7, 9, 11).FA cells from complementation groups B and E also express only theFANCD2-S. Functional correction of the MMC and IR sensitivity of theseFA cells with the corresponding FANC cDNA restored the FA proteincomplex (Garcia-Higuera et al., 1999) and restored the high molecularweight isoform (FANCD2-L) (FIG. 1A, lanes 4, 8, 10, 12). Taken together,these results demonstrate that the FA protein complex, containing FANCA,FANCC, FANCF, and FANCG, directly or indirectly regulates the expressionof the two isoforms of FANCD2. The six cloned FA genes therefore appearto interact in a common pathway.

Example 3 The FA Protein Complex is Required for the Monoubiquitinationof FANCD2

The high molecular weight isoform of FANCD2 could result from one ormore mechanisms, including alternative splicing of the FANCD2 mRNA orpost-translational modification(s) of the FANCD2 protein. Treatment withphosphatase did not convert FANCD2-L to FANCD2-S, demonstrating thatphosphorylation alone does not account for the observed difference intheir molecular mass.

In order to identify other possible post-translational modifications ofFANCD2, we initially sought cellular conditions which regulate theconversion of FANCD2-S to FANCD2-L (FIGS. 1B, C). Since FA cells aresensitive to MMC and IR, we reasoned that these agents might regulatethe conversion of FANCD2-S to FANCD2-L in normal cells. Interestingly,HeLa cells treated with MMC (FIG. 1B, lanes 1-6) or IR (FIG. 1C, lanes1-6) demonstrated a dose-dependent increase in the expression of theFANCD2-L isoform.

To determine whether FANCD2-L is a ubiquitinated isoform of FANCD2-S, wetransfected HeLa cells with a cDNA encoding HA-ubiquitin (Treier et al.,1994). Cellular exposure to MMC (FIG. 1B, lanes 7-10) or IR (FIG. 1C,lanes 7-10) resulted in a dose-dependent increase in the HA-ubiquitinconjugation of FANCD2. Only the FANCD2-L isoform, and not the FANCD2-Sisoform, was immunoreactive with an anti-HA antibody. Although FANCD2was not ubiquinated in FA cells, FANCD2 ubiquination was restored uponfunctional complementation of these cells. Although FANCD2 was notubiquitinated in FA cells, FANCD2 ubiquitination was restored uponfunctional complementation of these cells. Since the FANCD2-S andFANCD2-L isoforms differ by 7 kD, the FANCD2-L probably contains asingle ubiquitin moiety (76 amino acids) covalently bound by an amidelinkage to an internal lysine residue of FANCD2.

To confirm the monoubiquitination, we isolated FANCD2-L protein fromHeLa cells and analyzed its tryptic fragments by mass spectrometry (Wuet al., Science, (2000), Vol. 289, p. 11a). Ubiquitin tryptic fragmentswere unambiguously identified, and a site of monoubiquitination (K561 ofFANCD2) was also identified. Interestingly, this lysine residue isconserved among FANCD2 sequences from human, Drosophila, and C. elegans,suggesting that the ubiquitination of this site is critical to the FApathway in multiple organisms. Mutation of this lysine residue, FANCD2(K561R), resulted in loss of FANCD2 monoubiquitination.

Example 4 Formation of Nuclear Foci Containing FANCD2 Requires an IntactFA Pathway

We examined the immunofluorescence pattern of the FANCD2 protein inuncorrected, MMC-sensitive FA fibroblasts and functionally-complementedfibroblasts (FIG. 2).

The corrected FA cells expressed both the FANCD2-S and FANCD2-L isoforms(FIG. 2A, lanes 2, 4, 6, 8). The endogenous FANCD2 protein was observedexclusively in the nucleus of human cells, and no cytoplasmic stainingwas evident (FIG. 2B, a-h). The PD-20 (FA-D) cells have decreasednuclear immunofluorescence (FIG. 2B, d), consistent with the decreasedexpression of FANCD2 protein in these cells by immunoblot (FIG. 2A, lane7). In PD20 cells functionally-corrected with the FANCD2 gene bychromosome transfer, the FANCD2 protein stained in two nuclear patterns.Most corrected cells had a diffuse nuclear pattern of staining, and aminor fraction of cells stained for nuclear foci (see dots, panel h).Both nuclear patterns were observed with three independently-derivedanti-FANCD2 antisera (1 polyclonal, 2 monoclonal antisera). FAfibroblasts from subtypes A, G, and C showed only the diffuse pattern ofFANCD2 nuclear immunofluorescence. Functional complementation of thesecells with the FANCA, FANCG, or FANCC cDNA, respectively, restored theMMC resistance of these cells (Table 6), and restored the nuclear fociin some cells. The presence of the high molecular weight FANCD2-Lisoform therefore correlates with the presence of FANCD2 nuclear foci,suggesting that only the monoubiquitinated FANCD2-L isoform isselectively localized to these foci.

Example 5 The FANCD2 Protein is Localized to Nuclear Foci During S Phaseof the Cell Cycle

Since only a fraction of the asynchronous functionally-complementedcells contained FANCD2 nuclear foci, we reasoned that these foci mightassemble at discrete times during the cell cycle. To test thishypothesis, we examined the formation of the FANCD2-L isoform and FANCD2nuclear foci in synchronized cells (FIG. 3). HeLa cells weresynchronized at the G1/S boundary, released into S phase, and analyzedfor formation of the FANCD2-L isoform (FIG. 3A). The FANCD2-L isoformwas expressed specifically during late G1 phase and throughout S phase.Synchronized, uncomplemented FA cells (FA-A fibroblasts, GM6914)expressed normal to increased levels of FANCD2-S protein but failed toexpress FANCD2-L at any time during the cell cycle. Functionalcomplementation of these FA-A cells by stable transfection with theFANCA cDNA restored S phase-specific expression of FANCD2-L. The S phasespecific expression of the FANCD2-L isoform was confirmed when HeLacells were synchronized by other methods, such as nocodazole arrest(FIG. 3B) or mimosine exposure (FIG. 3B). Cells arrested in mitosis didnot express FANCD2-L, suggesting that the FANCD2-L isoform is removed ordegraded prior to cell division (FIG. 3B, mitosis). Taken together,these results demonstrate that the monoubiquitination of the FANCD2protein is highly regulated during the cell cycle, and that thismodification requires an intact FA pathway.

The cell cycle dependent expression of the FANCD2-L isoform alsocorrelated with the formation of FANCD2 nuclear foci (FIG. 3C).Nocodazole arrested (mitotic) cells express no FANCD2-L isoform andexhibit no FANCD2 nuclear foci (FIG. 3C, 0 hour). When thesesynchronized cells were allowed to traverse S phase (15 to 18 hours), anincrease in FANCD2 nuclear foci was observed.

Example 6 The FANCD2 Protein is Localized to Nuclear Foci in Response toDNA Damage

We examined the accumulation of the FANCD2-L isoform and FANCD2 nuclearfoci in response to DNA damage (FIG. 4). Previous studies have shownthat FA cells are sensitive to agents which cause DNA interstrandcrosslinks (MMC) or double strand breaks (IR) but are relativelyresistant to ultraviolet light (UV) and monofunctional alkylatingagents. MMC activated the conversion of FANCD2-S to FANCD2-L inasynchronous HeLa cells (FIG. 4A). Maximal conversion to FANCD2-Loccurred 12-24 hours after MMC exposure, correlating with the time ofmaximal FANCD2 nuclear focus formation. There was an increase in FANCD2nuclear foci corresponding to the increase in FANCD2-L. Ionizingradiation also activated a time-dependent and dose-dependent increase inFANCD2-L in HeLa cells, with a corresponding increase in FANCD2 foci(FIG. 4B). Surprisingly, ultraviolet (UV) light activated atime-dependent and dose-dependent conversion of FANCD2-S to FANCD2-L,with a corresponding increase in FANCD2 foci (FIG. 4C).

We tested the effect of DNA damage on FA cells (FIG. 4D). FA cells frommultiple complementation groups (A, C, and G) failed to activate theFANCD2-L isoform and failed to activate FANCD2 nuclear foci in responseto MMC or IR exposure. These data suggest that the cellular sensitivityof FA cells results, at least in part, from their failure to activateFANCD2-L and FANCD2 nuclear foci.

Example 7 Co-Localization of Activated FANCD2 and BRCA1 Protein

Like FANCD2, the breast cancer susceptibility protein, BRCA1, isupregulated in proliferating cells and is activated bypost-translational modifications during S phase or in response to DNAdamage. BRCA has a carboxy terminus 20 amino acids which contain ahighly acidic HMG-like domain suggesting a possible mechanism forchromatin repair. The BRCA1 protein co-localizes in IR-inducible foci(IRIFs) with other proteins implicated in DNA repair, such as RAD51 orthe NBS/Mre11/RAD50 complex. Cells with biallelic mutations in BRCA1have a defect in DNA repair and are sensitive to DNA damaging agentssuch as IR and MMC (Table 5). Taken together, these data suggest apossible functional interaction between the FANCD2 and BRCA1 proteins.BRCA foci are large (2mDa) multiprotein complexes including ATM and ATMsubstrates involved in DNA repair (BRCA1) and checkpoint functions(NBS).

In order to determine whether the activated FANCD2 protein co-localizeswith the BRCA1 protein, we performed double immunolabeling of HeLa cells(FIG. 5). In the absence of ionizing radiation, approximately 30-50% ofcells contained BRCA1 nuclear foci (FIG. 5A). In contrast, only rarecells traversing S phase contained FANCD2 dots (b, e). These nuclearfoci were also immunoreactive with antisera to both BRCA1 and FANCD2 (c,f). Following IR exposure, there was an increase in the number of cellscontaining nuclear foci and the number of foci per cell. These nuclearfoci were larger and more fluorescent than foci observed in the absenceof IR. Again, these foci contained both BRCA1 and FANCD2 protein (1,1).An interaction of FANCD2-L and BRCA1 was further confirmed bycoimmunoprecipitation of the proteins (FIG. 5B) from exponentiallygrowing HeLa cells exposed to IR.

We examined the effect of BRCA1 expression on the formation of FANCD2-Land nuclear foci (FIG. 6). The BRCA1 (−/−) cell line, HCC1937, expressesa mutant form of the BRCA1 protein with a carboxy terminal truncation.Although these cells expressed a low level of FANCD2-L (FIG. 6A), IRfailed to activate an increase in FANCD2-L levels. Also, these cells hada decreased number of IR-inducible FANCD2 foci (FIG. 6B, panels c, d).Correction of these BRCA1 (−/−) cells by stable transfection with theBRCA1 cDNA restored IR-inducible FANCD2 ubiquitination and nuclear foci(FIG. 6B, panels k, 1). These data suggest that the wild-type BRCA1protein is required as an “organizer” for IR-inducible FANCD2 dotformation and further suggests a functional interaction between theproteins.

Example 8 Co-Localization of FANCD2 and BRCA10n Meiotic Chromosomes

The association of FANCD2 and BRCA1 in mitotic cells suggested thatthese proteins might also co-localize during meiotic prophase. Previousstudies have demonstrated that the BRCA1 protein is concentrated on theunsynapsed/axial elements of human synaptonemal complexes in zygoteneand pachytene spermatocytes. To test for a possible colocalization ofFANCD2 and BRCA1 in meiotic cells, we examined surface spreads of latepachytene and early diplotene mouse spermatocytes for the presence ofFANCD2 and BRCA1 protein (FIG. 7). We found that the rabbit polyclonalanti-FANCD2 antibody E35 specifically stained the unpaired axes of the Xand Y chromosomes in late pachynema (FIG. 7 a) and in diplonema (FIGS. 7d, 7 e and 7 g). Under the same experimental conditions, preimmune serumdid not stain synaptonemal complexes (FIGS. 7 b and 7 c). The M118anti-BRCA1 antibody stained the unpaired sex chromosomes in mousepachytene and diplotene spermatocytes (FIGS. 7 f and 7 h). FANCD2 Abstaining of the unsynapsed axes of the sex chromosomes was interrupted,giving a beads-on-a-string appearance (FIG. 7 g). A consecutiveexamination of 20 pachytene nuclei indicated that most (˜65%) of theseanti-FANCD2 foci co-localized with regions of intense anti-BRCA1staining, further supporting an interaction between these proteins(FIGS. 7 g, 7 h, and 7 i). These results provide the first example of aFANC protein (activated FANCD2) which binds to chromatin.

Example 9 Experimental Protocols for Obtaining and Analyzing the DNA andProtein Sequence for FANCD2

Northern Hybridizations. Human adult and fetal multi-tissue mRNA blotswere purchased from Clontech (Palo Alto, Calif.). Blots were probed with32P labeled DNA from EST clone SGC34603. Standard hybridization andwashing conditions were used. Equal loading was confirmed byre-hybridizing the blot with an actin cDNA probe.

Mutation Analysis. Total cellular RNA was reverse transcribed using acommercial kit (Gibco/BRL). The 5′ end section of FANCD2 was amplifiedfrom the resulting patient and control cDNA with a nested PCR protocol.The first round was performed with primers (SEQ ID NO:97) MG4715′-AATCGAAAACTACGGGCG-3′ and (SEQ ID NO:98) MG4575′-GAGAACACATGAATGAACGC-3′. The PCR product from this round was diluted1:50 for a subsequent round using primers (SEQ ID NO:99) MG4925′-GGCGACGGCTTCTCGG AAGTAATTTAAG-3′ and (SEQ ID NO:100) MG4725′-AGCGGCAGGAGGTTTATG-3′. The PCR conditions were as follows: 94° C. for3 min, 25 cycles of 94° C. for 45 sec, 50° C. for 45 sec, 72° C. for 3min and 5 min of 72° C. at the end. The 3′ portion of the gene wasamplified as described above but with primers, (SEQ ID NO:101) MG4745′-TGGCGGCAGACAGAAG TG-3′ and (SEQ ID NO:102) MG4755′TGGCGGCAGACAGAAGTG-3′. The second round of PCR was performed with (SEQID NO:103) MG491 5′-AGAGAGCCAACCTGAGCGA TG-3′ and (SEQ ID NO:104) MG4765′-GTGCCAGACTCTGGTGGG-3′. The PCR products were gel-purified, clonedinto the pT-Adv vector (Clontech) and sequenced using internal primers.

Allele specific assays. Allele specific assays were performed in thePD20 family and 290 control samples (=580 chromosomes). The PD20 familyis of mixed Northern European descent and VU008 is a Dutch family.Control DNA samples were from unrelated individuals in CEPH families(n=95), samples from unrelated North American families with eitherectodermal dysplasia (n=95) or Fanconi Anemia (n=94). The maternalnt376a→g mutation in the PD20 family created a novel MspI restrictionsite. For genomic DNA, the assay involved amplifying genomic DNA usingthe primers (SEQ ID NO:105) MG792 5′-AGGAGACACCCTTCCTATCC-3′ located inexon 4 and (SEQ ID NO:106) M0803 5′-GAAGTTGGCAAAACAGAC TG-3′ which is inintron 5. The size of the PCR product was 340 bp, yielding two fragmentsof 283 bp and 57 bp upon Mspl digestion if the mutation was present. Foranalysis of the reverted cDNA clones, PCR was performed using primers(SEQ ID NO:107) MG924 5′-TGTCTTGTGA GCGTCTGCAGG-3′ and (SEQ ID NO:108)MG753 5′-AGGTT TTGATAATGGCAGGC-3′. The paternal exon 37 mutation(R1236H) in PD20 and exon 12 missense mutation (R302W) in VU008 weretested by allele specific oligonucleotide (ASO) hybridization (Wu etal., DNA, (1989) Vol. 8, pp. 135-142). For the exon 12 assay, genomicDNA was amplified with primers (SEQ ID NO:109) MG9795′-ACTGGACTGTGCCTACCCACTATG-3′ and (SEQ ID NO:110) MG9845′-CCTGTGTGAGGATGAGCTCT-3′. Primers (SEQ ID NO:171) MG8185′-AGAGGTAGGGAAGGAAGCTAC-3′ and (SEQ ID NO:172) MG813 5′-CCAAAGTCCACTTCTTGAAG-3′ were used for exon 37. Wild-type (SEQ ID NO:111)(5′-TTCTCCCGAAG CTCAG-3′ for R302W and (SEQ ID NO:112)5′-TTTCTTCCGTGTGATGA-3′ for R1236H and mutant SEQ ID NO:351(5′-TTCTCCCAAAGCTGAG-3′ R302W and SEQ ID NO:352 (5′-TYTCTTCCATGTGATGA-3′for R1236H) oligonucleotides were end-labeled with γ32P-[ATP] andhybridized to dot-blotted target PCR products as previously ss novelDdeI site. The wild-type PCR product digests into a 117 and 71 bpproduct, whereas the mutant allele yields three fragments of 56, 61 and71 bps in length. PCR in all of the above assays was performed with 50ng of genomic DNA for 37 cycles of 94° C. for 25 sec, 50° C. for 25 secand 72° C. for 35 sec.

Generation of an anti-FANCD2 antiserum. A rabbit polyclonal antiserumagainst FANCD2 was generated using a GST-FANCD2 (N-terminal) fusionprotein as an antigen source. A 5′ fragment was amplified by polymerasechain reaction (PCR) from the full length FANCD2 cDNA with the primers(SEQ ID NO:113) DF4EcoRJ (5′-AGCCTCgaattcGUTCC AAAAGAAGACTGTCA-3′) and(SEQ ID NO:114) DR816Xh (5′-GGTATCctcgagTCAAGA CGACAACTTATCCATCA-3′).The resulting PCR product of 841 bp, encoding the amino-terminal 272amino acids of the FANCD2 polypeptide was digested with EcoRI/XhoI andsubcloned into the EcoRI/XhoI sites of the plasmid pGEX4T-1 (Pharmacia).A GST-FANCD2 (N-terminal) fusion protein of the expected size (54 kD)was expressed in E. coli strain DH5α, purified overglutathione-S-sepharose, and used to immunize a New Zealand Whiterabbit. An FANCD2-specific immune antiserum was affinity-purified overan AminoLink Plus column (Pierce) loaded with GST protein and over anAminoLink Plus column loaded with the GST-FANCD2 (N-terminal) fusionprotein.

Immunoblotting is as in Example 1.

Cell Lines and Transfections. PD20i is an immortalized and PD733 aprimary FA fibroblast cell line generated by the Oregon Health SciencesFanconi Anemia cell repository (Jakobs et al., Somet. Cell. Mol. Genet.,(1996), Vol. 22, pp. 151-157). PD20 lymphoblasts were derived from bonemarrow samples. VU008 is a lymphoblast and VU423 a fibroblast linegenerated by the European Fanconi Anemia Registry (EUFAR). VU423i was animmortalized line derived by transfection with SV40 T-antigen (Jakobs etal., 1996) and telomerase (Bodnar et al., Science, (1998) Vol. 279, pp.349-352). The other FA cell lines have been previously described. Humanfibroblasts were cultured in MEM and 20% fetal calf serum. Transformedlymphoblasts were cultured in RPMI 1640 supplemented with 15%heat-inactivated fetal calf serum.

To generate FANCD2 expression constructs, the full-length cDNA wasassembled from cloned RT-PCR products in pBluescript and the absence ofPCR induced mutations was confirmed by sequencing. The expressionvectors pIRES-Neo, pEGFP-N1, pRevTRE and pRevTet-off were from ClonTech(Palo Alto, Calif.). The FANCD2 was inserted into the appropriatemulti-cloning site of these vectors. Expression constructs wereelectroporated into cell line PD20 and a normal control fibroblast cellline, GM639 using standard conditions (van den Hoff et al., 1992).Neomycin selection was carried out with 400 μg/ml active G418 (Gibco).

Whole cell fusions. For the whole cell fusion experiments, a PD20 cellline (PD20i) resistant to hygromycin B and deleted for the HPRT locuswas used (Jakobs et al., Somat. Cell. Mol. Genet., (1997) Vol. 23, pp.1-7). Controls included PD24 (primary fibroblasts from affected siblingof PD20) and PD319i (Jakobs et al., 1997) (immortal fibroblasts from anon-A, C, D or G FA patient). 2.5×105 cells from each cell line weremixed in a T25 flask and allowed to recover for 24 hours. The cells werewashed with serum-free medium and then fused with 50% PEG for 1 min.After removal of the PEG, the cells were washed 3× with serum-freemedium and allowed to recover overnight in complete medium withoutselection. The next day, cells were split 1:10 into selective mediumcontaining 400 μg/ml hygromycin B (Roche Molecular) and 1×HAT. After theselection was complete, hybrids were passaged once and then analyzed asdescribed below.

Retroviral Transduction of FA-D2 cells and complementation analysis. Thefull length FANCD2 cDNA was subcloned into the vector, pMMP-puro(Pulsipher et al., 1998). Retroviral supernatants were used to transducePD20F, and puromycin resistant cells were selected. Cells were analyzedfor MMC sensitivity by the crystal violet assay (Naf et al., 1998).

Chromosome Breakage Analysis. Chromosome breakage analysis was performedby the Cytogenetics Core Lab at OHSU (Portland, Oreg.). For the analysis(Cohen et al., 1982) cells were plated into T25 flasks, allowed torecover and then treated with 300 ng/ml of DEB for two days. Aftertreatment, the cells were exposed to colcemid for 3 hours and harvestedusing 0.075 M KCl and 3:1 methanol:acetic acid. Slides were stained withWright's stain and 50-100 metaphases were scored for radials.

Example 10 Mouse Models for FA for Use in Screening PotentialTherapeutic Agents

Murine models of FANCD2 can be made using homologous recombination inembryonic stem cells or targeted disruption as described in D'Andrea etal., (1997) 90:1725-1736, and Yang et al., Blood, (2001) Vol. 98, pp.1-6. The knockout of FANCD2 locus in mice is not a lethal mutation.These knock-out animals have increased susceptibility to cancer andfurthermore display other symptoms characteristic of FA. It is expectedthat administering certain therapeutic agents to the knock-out mice willreduce their susceptibility to cancer. Moreover, it is expected thatcertain established chemotherapeutic agents will be identified that aremore effective for treating knock-out mice who have developed cancers asa result of the particular genetic defect and this will also be usefulin treating human subjects with susceptibility to cancer or who havedeveloped cancers as a result of a mutation in the FANCD2 locus.

We can generate experimental mice models with targeted disruptions ofFANCD2 using for example the approach described by Chen et al, Nat.Genet., (1996) Vol. 12, pp. 448-451, for FANCC who created a disruptionin an exon of the gene, and by Whitney et al., (1996) Vol. 88, pp.49-58, who used homologous recombination to create a disruption of anexon of the gene. In both animal models, spontaneous chromosome breakageand an increase in chromosome breaks in splenic lymphocytes in responseto bifunctional alkylating agents are observed. In both models,FANCD2−/− mice have germ cell defects and decreased fertility. TheFANCD2 murine knockout model is useful in examining (1) the role of theFANCD2 gene in the physiologic response of hematopoietic cells to DNAdamage, (2) the in vivo effects of inhibitory cytokines on FA marrowcells, and (3) the efficacy of gene therapy and (4) for screeningcandidate therapeutic molecules.

The availability of other FA gene disruptions will allow the generationand characterization of mice with multiple FA gene knockouts. Forinstance, if 2 FA genes function exclusively in the same cellularpathway, a double knockout should have the same phenotype as the singleFA gene knockout.

The murine FANCD2 gene can be disrupted by replacing exons with anFRT-flanked neomycin cassette via homologous recombination in 129/SvJaeembryonic stem cells. Mice homozygous for the FANCD2 mutation within amixed genetic background of 129/Sv and C57BL can be generated followingstandard protocols. Mouse tail genomic DMA can be prepared as previouslydescribed and used as a template for polymerase chain reaction (PCR)genotyping.

Splenocytes can be prepared from 6-week-old mice of known FANCD2genotype. The spleen is dissected, crushed in RPMI medium into asingle-cell suspension, and filtered through a 70 μm filter. Red cellsare lysed in hypotonic ammonium chloride. The remaining spleniclymphocytes are washed in phosphate-buffered saline and resuspended inRPMI/10% fetal bovine serum plus phytohemagglutinin. Cells are testedfor viability by the trypan blue exclusion assay. Cells are cultured for24 hours in media and exposed to MMC or DEB for an additional 48 hours.Alternatively, cells are cultured for 50 hours, exposed to IR (2 or 4Gy, as indicated), and allowed to recover for 12 hours before chromosomebreakage or trypan blue exclusion (viability) analysis.

Mononuclear cells can be isolated from the femurs and tibiae of 4- to6-week-old FANCD2+/− or FANCD2−/− mice, as previously described. A totalof 2×104 cells were cultured in 1 mL of MethoCult M343 media (StemCellTechnologies, Vancouver, BC) with or without MMC treatment. Colonies arescored at day 7, when most of the colonies belong to thegranulocyte-macrophage colony-forming unit or erythroid burst-formingunit lineages. Each number are averaged from duplicate plates, and thedata derived from 2 independent experiments.

Lymphocytes isolated from thymus, spleen, and peripheral lymph nodes arestained for T- or B-lymphocyte surface molecules with fluoresceinisothiocyanate-conjugated anti-CD3, CD4, and CD19 and PE-conjugatedanti-CD8, CD44, CD 45B, immunoglobulin M, and B220 (BD PharMingen,Calif.). Stained cells were analyzed on a Counter Epics XL flowcytometry system.

Mice ovaries and testes were isolated and fixed in 4% paraformaldehydeand further processed by the core facility of the Department ofPathology at Massachusetts General Hospital.

Example 11 Screening Assays Using Antibody Reagents for DetectingIncreased Cancer Susceptibility in Human Subjects

Blood samples or tissue samples can be taken from subjects for testingfor the relative amounts of FANCD2-S compared to FANCD2-L and thepresence or absence of FANCD2-L. Using antibody reagents specific forFANCD2-S and FANCD2-L proteins (Example 1), positive samples can beidentified on Western blots as shown in FIG. 14. Other antibody assaysmay be utilized such as, for example, one step migration binding bandedassays described in U.S. Pat. Nos. 5,654,162 and 5,073,484. Enzymelinked immunosorbent assays (ELISA), sandwich assays, radioimmune assaysand other immunodiagnostic assays known in the art may be used todetermine relative binding concentrations of FANCD2-S and FANCD2-L.

The feasibility of this approach is illustrated by the following:

FANCD2 Diagnostic Western Blot for Screening Human Cancer Cell Lines

Human cancer cell lines were treated with or without ionizing radiation(as indicated in FIG. 14) and total cell proteins were electrophoresed,transferred to nitrocellulose and immunoblotted with the anti-FANCD2monoclonal antibody of Example 1. Ovarian cancer cell line (TOV21G)expressed FANCD2-S but not FANCD2-L (see lanes 9, 10). This cell linehas a deletion of human chromosome 3p overlapping the FANCD2 gene and ishemizygous for FANCD2 and is predicted to have a mutation in the secondFANCD2 allele which therefore fails to be monoubiquinated by the PAcomplex hence no FANCD2-L (lanes 9, 10). This example demonstrates thatantibody based tests are suited for determining lesions in the FANCD2gene which lead to increased cancer susceptibility.

Example 12 Screening Assays Using Nucleic Acid Reagents for DetectingIncreased Cancer Susceptibility in Human Subjects

Blood samples or tissue samples can be taken from subjects and screenedusing sequencing techniques or nucleic acid probes to determine the sizeand location of the genetic lesion if any in the genome of the subject.The screening method may include sequencing the entire gene or by usingsets of probes or single probes to identify lesions. It is expected thata single lesion may predominant in the population but that other lesionsmay arise throughout the gene with low frequency as is the case forother genetic conditions such as cystic fibrosis and the P53 tumorsuppressor gene.

The feasibility of this approach is illustrated by the following:

Peripheral blood lymphocytes are isolated from the patient usingstandard Ficoll-Hypaque gradients and genomic DNA is isolated from theselymphocytes. We use genomic PCR to amplify 44 exons of the human FANCD2gene (see primer Table 7) and sequence the two FANCD2 alleles toidentify mutations. Where such mutations are found, we distinguish thesefrom benign polymorphisms by their ability to ablate the functionalcomplementation of an FA-D2 indicator cell line.

Example 13 Measurement of Mono-Ubiquitinated FANC D2-L in TissueBiopsies

Tissue biopsies were obtained by needle aspiration or skin punch biopsy.Cells, resuspended in appropriate culture media in microtiter plates arethen treated with the indicated concentration of MMC (0, 10, 40, 160ng/ml) or the indicated dose of IR (0, 5, 10, 10, 20 Gy). After 24hour-incubation with MMC, or two hours after IR treatment, whole cellextracts were prepared in Lysis Buffer (50 mM TrisHCl pH 7.4, 150 mMNaCl, 1% (v/v) Triton X-100) supplemented with protease inhibitors (1μg/ml leupeptin and pepstatin, 2 μg/ml aprotinin, 1 mMphenylmethylsulfonylfluoride) and phosphatase inhibitors (1 mM sodiumorthovanadate, 10 mM sodium fluoride). Samples are then tested for thepresence of the FANC D2-L isoform using the anti-FANCD2-L-specificmonoclonal antibody, as disclosed herein, and conventional immunoassayssuch as the enzyme linked immunosorbent assay (ELISA) that are commonlyused to quantitate the levels of proteins in cell samples (see Harlow,E. and Lane, D. Using Antibodies: A Laboratory Manual (1999) Cold SpringHarbor Laboratory Press).

Example 14 Diagnosis of Cancer Associated Defects in a FanconiAnemia/BRCA Gene or Protein

PCR Amplification and Sequencing of the Human FANCD2 Gene—cDNA andGenomic DNA Templates

Genomic DNA Sequencing

In the course of sequencing the FANCD2 gene, it became apparent thatthere are at least eight pseudogene sequences for FANCD2 in the humangenome, all located on human chromosome 3p (see attached Table 8).Accordingly, it was important to design a specific genomic PCR assay,designed to specifically amplify the FANCD2 sequence and to exclude thepseudogenes. It is not possible to design PCR primers close to exons 1,2, 3, 7-14, 19-22, 23-29, 30-32, 33-36 and 43-44 of the functionalFANCD2 gene that do not also amplify one or more of the non-functionalcopies of those exons. By first generating large PCR products that areunique to these regions of the functional gene, then using those uniqueproducts as templates in subsequent amplification reactions to produceexonic PCR products with primers that are not unique to the functionalgene, a vast excess of the PCR products from the functional gene overthe PCR products from the copies was generated. In this manner,mutations in the functional gene are made detectable.

Superamplicon PCR

As indicated above, the purpose of these PCR reactions is to generatelarge amplicons (superamplicons) that are unique to certain regions ofthe functional FANCD2 gene. The components of the PCR are: 60 mMTris-SO₄ (pH8.9), 18 mM (NH₄)2SO₄, 2.0 mM MgSO₄, 0.2 mM in each of dATP,dCTP, dGTP, TTP, 0.1 μM of each primer, 5 ng/μl DNA, 0.05 units/μlPlatinum Taq DNA Polymerase High Fidelity (GIBCO BRL, Gaithersburg,Md.).

The thermocycling conditions are: 94° C., 4 min, followed by 11 cycles,each with a denaturing step at 94° C. for 20 seconds and an extensionstep at 72° C. for 300 seconds, and with a 20 second annealing step thatdecreased 1° C./cycle, beginning at 64° C. in the first cycle anddecreasing to 54° C. in the eleventh cycle; the eleventh cycle was thenrepeated 25 times; a 6 minute incubation at 72° C. followed by a 4° C.soak completed the program.

The primer identities are as follows (the primer sequences are in thetable 9):

Amplicon Amplicon Exons FwdPrimer RevPrimer Length Name x1-x2 exon 2 Fsuper-1-2 R 2097  1 super exon 1 F super-1-2 R 4346  2 super x3super-3-F exon 3 R 2323  3 super x7-x14 exon-10-F super-7-14-R 5635  4super super-7-14-F exon-9-R 4595  5 super x19-x22 exon-21-F super-19-22R 1015  6 super super-19-22-F exon-20-R 2749  7 super x23-x29 exon-27 Fsuper-23-29 R 3371  9 super super-23-29 F exon 26 R 3252 10 superx30-x32 exon 31 F super-30-32 R 2895 11 super super-30-32 F exon 30 R299 12 super x33-36 exon 35 F super-33-36 R 2186 13 super super-33-36 Fexon 34 R 3457 14 super x43-x44 exon 44 F super-43-44 R 464 15 supersuper-43-44 F exon 43a R 2040 16 super

Exonic PCR

These PCR's are of 2 types: (1) the superamplicon PCR is used as the DNAtemplate; exons 1-3, 7-14, 19-22, 23-29, 30-32, 33-36 and 43-44 are inthis group, and (2) unamplified genomic DNA is used as the DNA template;exons 4-6, 15-18 and 37-42 are in this group.

One primer (designated “-F”) in each pair was synthesized with an 18base M13-21 forward sequence (SEQ ID NO: 192)(TGTAAAACGACGGCCAGT) at its5′ end, and the other primer (designated “-R”) was synthesized with an18 base M13-28 reverse sequence (SEQ ID NO: 193) (CAGGAAACAGCTATGACC) atits 5′ end. For exon 15, two overlapping amplicons were designed.

The components of the 10 ul PCR reaction are: 20 mM Tris-HCl(pH8.4), 50mM KCl, 1.5 mM MgCl₂, 0.1 mM in each of dATP, dCTP, dGTP, TTP, 0.1 μM ofeach primer, either 1 ul of a 1:100 dilution of the superamplicon PCR or5 ng/ul of unamplified genomic DNA, 0.05 units/μl Taq polymerase (TaqPlatinum, GIBCO BRL, Gaithersburg, Md.). The thermocycling conditionsare: 94° C., 4 min, followed by 11 cycles, each with a denaturing stepat 94° C. for 30 seconds and an extension step at 72° C. for 20 seconds,and with a 20 second annealing step that decreased 1° C./cycle,beginning at 60° C. in the first cycle and decreasing to 50° C. in theeleventh cycle; the eleventh cycle was then repeated 25 times; a 6minute incubation at 72° C. followed by a 4° C. soak completed theprogram.

cDNA Sequencing

Two micrograms of total RNA is converted into cDNA using SuperscriptFirst-Strand Synthesis System for RT-PCR (GIBCO/BRL) according to themanufacturer's instructions. One twentieth of the RT-PCR reaction isused as the DNA template in each of 18 PCR reactions; these PCRreactions amplify the coding region of the cDNA in overlappingfragments. The primers are shown in the table below.

One primer (designated “-F”) in each pair was synthesized with an 18base M13-21 forward sequence (TGTAAAACGACGGCCAGT) at its 5′ end, and theother primer (designated “-R”) was synthesized with an 18 base M13-28reverse sequence(CAGGAAACAGCTATGACC) at its 5′ end.

The components of the 10 ul PCR reaction are: 20 mM Tris-HCl(pH8.4), 50mM KCl, 1.5 mM MgCl₂, 0.1 mM in each of dATP, dCTP, dGTP, TTP, 0.1 μM ofeach primer, either 1 ul of a 1:100 dilution of the superamplicon PCR or5 ng/μl of unamplified genomic DNA, 0.05 units/μl Taq polymerase (TaqPlatinum, GIBCO BRL, Gaithersburg, Md.).

The thermocycling conditions are: 94° C., 4 min, followed by 11 cycles,each with a denaturing step at 94° C. for 30 seconds and an extensionstep at 72° C. for 20 seconds, and with a 20 second annealing step thatdecreased 1° C./cycle, beginning at 60° C. in the first cycle anddecreasing to 50° C. in the eleventh cycle; the eleventh cycle was thenrepeated 25 times; a 6 minute incubation at 72° C. followed by a 4° C.soak completed the program.

Primer 5′ Position Sequence (5′ to 3′) Length (bp) D1F   24TGTAAAACGACGGCCAGT CGACGGCTTCTCGGAAGTAA SEQ ID NO: 194 D1R  408AGGAAACAGCTATGACCAT GCAGACGCTCACAAGACAAA 407 SEQ ID NO: 195 D2F  322TGTAAAACGACGGCCAGT GACACCCTTCCTATCCCAAAA SEQ ID NO: 196 D2R  689AGGAAACAGCTATGACCAT CAGGTTCTCTGGAGCAATAC 368 SEQ ID NO: 197 D3F  612TGTAAAACGACGGCCAGT TGGCTTGACAGAGTTGTGGAT SEQ ID NO: 198 D3R 1019AGGAAACAGCTATGACCAT CTGTAACCGTGATGGCAAAAC 408 SEQ ID NO: 199 D4F  855TGTAAAACGACGGCCAGT CGCCAGTTGGTGATGGATAAG SEQ ID NO: 200 D4R 1223AGGAAACAGCTATGACCAT AAGCATCACCAGGTCAAACAC 369 SEQ ID NO: 201 D5F 1081TGTAAAACGACGGCCAGT GCGGTCAGAGCTGTATTATTC SEQ ID NO: 202 D5R 1461AGGAAACAGCTATGACCAT CTGCTGGCAGTACGTGTCAA 401 SEQ ID NO: 203 D6F 1377TGTAAAACGACGGCCAGT TCGCTGGCTCAGAGTTTGCTT SEQ ID NO: 204 D6R 1765AGGAAACAGCTATGACCAT GTGCTAGAGAGCTGCTTTCTT 389 SEQ ID NO: 205 D7F 1641TGTAAAACGACGGCCAGT CCCCTCAGCAAATACGAAAAC SEQ ID NO: 206 D7R 2065AGGAAACAGCTATGACCAT ACTACGAAGGCATCCTGGAAA 424 SEQ ID NO: 207 D8F 1947TGTAAAACGACGGCCAGT GCCTCTGCACTTTACTATGATG SEQ ID NO: 208 D8R 2301AGGAAACAGCTATGACCAT CTCCTCCAAGTTTCCGTTATG 375 SEQ ID NO: 209 D9F 2210TGTAAAACGACGGCCAGT GGTGACCTCACAGGAATCAG SEQ ID NO: 210 D9R 2573AGGAAACAGCTATGACCAT TTTCCAAGAGGAGGGACATAG 384 SEQ ID NO: 211 D10F 2438TGTAAAACGACGGCCAGT CAACTGGTTCCGAGAGATTGT SEQ ID NO: 212 D10R 2859AGGAAACAGCTATGACCAT CAATGTCCAGCTCTCGGAAAAA 422 SEQ ID NO: 213 D11F 2746TGTAAAACGACGGCCAGT GTGACCCTACGCCATCTCATA SEQ ID NO: 214 D11R 3138AGGAAACAGCTATGACCAT ACATTGGGGTCAGCAGTTGAA 393 SEQ ID NO: 215 D12F 3027TGTAAAACGACGGCCAGT AGAGTCCCCTTTCTCAAGAACA SEQ ID NO: 216 D12R 3413AGGAAACAGCTATGACCAT GACGCTCTGGCTGAGTAGTT 387 SEQ ID NO: 217 D13F 3334TGTAAAACGACGGCCAGT CAGCCCTCCATGTCCTTAGT SEQ ID NO: 218 D13R 3742AGGAAACAGCTATGACCAT AGGGAATGTGGAGGAAGATG 407 SEQ ID NO: 219 D14F 3637TGTAAAACGACGGCCAGT TGGAGCACACAGAGAGCATT SEQ ID NO: 220 D14R 4010AGGAAACAGCTATGACCAT GTCTAGGAGCGGCATACATT 374 SEQ ID NO: 221 D15F 3830TGTAAAACGACGGCCAGT AGCAGACTCGCAGCAGATTCA SEQ ID NO: 222 D15R 4225AGGAAACAGCTATGACCAT AGCCAGAAAGCCTCTCTACA 396 SEQ ID NO: 223 D16F4117/4112 TGTAAAACGACGGCCAGT ACACGAGACTCACCCAACAT SEQ ID NO: 224 D16R-L4477 AGGAAACAGCTATGACCAT GGGAATGGAAATGGGCATAGA 361 SEQ ID NO: 225 D16R-S4451 AGGAAACAGCTATGACCAT GACACAGAAGCAGGCAACAA 340 SEQ ID NO: 226D17F-(L) 4333 TGTAAAACGAGGGCCAGT AGAGCAAAGCCACTGAGGTAT SEQ ID NO: 227D17R-(L) 4768 AGGAAACAGCTATGACCAT GACTCTGTGCTTTGGCTTTCA 436SEQ ID NO: 228

DNA Sequencing

An aliquot of each PCR reaction was diluted 1:10 with water. The dilutedPCR product was sequenced on both strands using an M13 Forward and anM13 Reverse Big Dye Primer kit (Applied Biosystems, Foster City, Calif.)according to the manufacturer's recommendations. The sequencing productswere separated on a fluorescent sequencer (model 377 from AppliedBiosystems, Foster City, Calif.). Base calls were made by the instrumentsoftware, and reviewed by visual inspection. Each sequence was comparedto the corresponding normal sequence using Sequencher 3.0 software(LifeCodes).

Example 15 Method of Screening for a Chemosensitizing Agent

As shown in the model of the FA/BRCA pathway, the enzymaticmonoubiquitination of FANCD2 is a critical regulatory event. This eventrequires an intact FA protein complex (A/C/E/F/G complex) and requiresBRCA1 and BRCA2. While the actual catalytic subunit required for FANCD2monoubiquitination remains unknown, it still remains possible to screenfor antagonists of monoubiquitination. As described elsewhere in thistext, an inhibitor of the FA pathway could, in principal, function as achemosensitizer of cisplatin in the treatment of ovarian cancer or othercancers. The screening of an inhibitor of FANCD2 monoubiquitination canbe performed as a simple mammalian cell-based screen. A mammalian tissueculture cell line, e.g., Hela calls are first preincubated with randomcandidate small molecules. Cell clones are then screened usinganti-FANCD2 western blots. An inhibitor (antagonist) of the FA pathwaywill block FANCD2 monoubiquitination.

As described in Garcia-Higuera et al, 2001, BRCA1 may in fact be theenzyme which monoubiquitinates FANCD2. Accordingly, BRCA1 has aubiquitin ligase (Ring Finger) catalytic domain. Therefore, an in vitroassay will be devised to screen for BRCA1-mediated monoubiquitination ofFANCD2. An inhibitor will be screened directly for its ability toinhibit this in vitro reaction. Once inhibitors are identified, suchdrugs could be used in animal studies or phase 1 human studies todetermine their functions as cisplatin sensitizers.

Example 16 Method of Screening for a Potential Cancer Therapeutic

Cells and animals which carry a Fanconi Anemia/BRCA pathway gene havingone or more cancer associated defects can be used as model systems tostudy and test for substances which have potential as therapeuticagents. The cells are typically cultured epithelial cells. These may beisolated from individuals with Fanconi Anemia/BRCA pathway gene havingone or more cancer associated defects, either somatic or germline.Alternatively, the cell line can be engineered to carry the mutation ina gene of the Fanconi Anemia/BRCA pathway gene having one or more cancerassociated defects.

After a test substance is applied to the cells, the neoplasticallytransformed phenotype of the cell is determined. Any trait ofneoplastically transformed cells can be assessed, includinganchorage-independent growth, tumorigenicity in nude mice, invasivenessof cells, and growth factor dependence. Assays for each of these traitsare known in the art.

Animals for testing therapeutic agents can be selected after mutagenesisof whole animals or after treatment of germline cells or zygotes. Suchtreatments include insertion of mutant Fanconi Anemia/BRCA pathway geneshaving one or more cancer associated defects, usually from a secondanimal species, as well as insertion of disrupted homologous genes.Alternatively, the endogenous Fanconi Anemia/BRCA pathway gene(s) of theanimals may be disrupted by insertion or deletion mutation or othergenetic alterations using conventional techniques (Capecchi, 1989;Valancius and Smithies, 1991; Hasty et al., 1991; Shinkai et al., 1992;Mombaerts et al., 1992; Philpott et al., 1992; Snouwaert et al., 1992;Donehower et al., 1992) as outlined in Example 10. After test substanceshave been administered to the animals, the growth of tumors must beassessed. If the test substance prevents or suppresses the growth oftumors, then the test substance is a candidate therapeutic agent for thetreatment of the cancers identified herein.

Example 17 Method of Treatment of a Cancer that is Resistant to anAnti-Neoplastic Agent

The present example describes the treatment of a patient with a cancerthat is resistant to an anti-neoplastic agent such as cisplatin. Theprotocol provides for the administration of cisplatin as describedherein with an increasing dosage of an inhibitor of the ubiquitinationof the FANC D2 protein as a chemosensitizing agent. Cisplatin and thechemosensitizing agent can be administered intravenously,subcutaneously, intratumorally or intraperitoneally. The administeringphysician can adjust the amount and timing of drug administration on thebasis of results observed using standard measures of cancer activityknown in the art. Suppression of tumor growth and metastasis isindicative of effective treatment of the cancer.

Example 18 A Method of Measuring the Future Efficacy of a TherapeuticAgent

Tissue biopsies of neoplasms from cancer patients being treated with atherapeutic agent are obtained by needle aspiration or skin punchbiopsy. Cells, resuspended in appropriate culture media in microtiterplates are then treated with the indicated concentration of MMC (0, 10,40, 160 ng/ml) or the indicated dose of IR (0, 5, 10, 10, 20 Gy). After24 hour-incubation with MMC, or two hours after IR treatment, it induceDNA damage, whole cell extracts were prepared in Lysis Buffer (50 mMTrisHCl pH 7.4, 150 mM NaCl, 1% (v/v) Triton X-100) supplemented withprotease inhibitors (1 μg/ml leupeptin and pepstatin, 2 μg/ml aprotinin,1 mM phenylmethylsulfonylfluoride) and phosphatase inhibitors (1 mMsodium orthovanadate, 10 mM sodium fluoride). Samples are then testedfor the presence of the FANC D2-L isoform using theanti-FANCD2-L-specific monoclonal antibody, as disclosed herein, andconventional immunoassays such as the enzyme linked immunosorbent assay(ELISA) that are commonly used to quantitate the levels of proteins incell samples (see Harlow, E. and Lane, D. Using Antibodies: A LaboratoryManual (1999) Cold Spring Harbor Laboratory Press). Detection of themono-ubiquitinated FANC D2-L isoform is considered indicative of areduced efficacy of the therapeutic agent being used to treat the cancerpatient.

Example 19 A Method of Determining Resistance to a Chemotherapy Agent

A flow chart describing the protocol used to determine the methylationstate of the Fanconi Anemia/BRCA pathway genes is depicted in FIG. 21.

Analysis of FANCF Methylation.

DNA methylation patterns in FANCF gene were determined by methylationspecific PCR or PCR-based HpaII restriction enzyme assay. Genomic DNAwas isolated from indicated cell lines using QIAamp DNA Blood Mini Kit(QIAGEN).

PCR-Based HpaII Restriction Enzyme Assay

250 ng of genomic DNA was digested with 30 unit of HpaII or MspI for 12hr at 37° C. 12.5 ng of DNA from each digest was analyzed by PCR in 10μl reactions containing 1×PCR buffer, 200 μM each of the fourdeoxynucleotide triphosphates, 0.5 units of AmpliTaq DNA polymerase(Roche), and 0.2 μM of each primer. PCR was run for 33 cycles, and eachcycle constituted denaturation (45 sec at 94° C., first cycle 4 min 45sec), annealing (1 min at 61 20° C.), and extension (2 min at 72° C.,last cycle 9 min) PCR reaction was subjected to electrophoresis on a1.2% agarose gel containing ethidium bromide. Primers used were (SEQ IDNO:229) FPF6 (5′-GCACCTCATGGAATCCCTTC-3′)(forward) and (SEQ ID NO:230)FR343 (5′-GTTGCTGCACCAGGTGGTAA-3′)(reverse). These primers were designedusing nt -6-14 for the forward primer and nt 403-432 for the reverseprimer.

Methylation-Specific PCR.

Bisulfite modification of genomic DNA was performed as previouslydescribed (Herman J G et al. Proc Natl Acad Sci USA 93 (18) 9821-6(1996)). The bisuffite-treated DNA was amplified with either amethylation-specific or unmethylation-specific primer set. PCR was runfor 40 cycles, and each cycle constituted denaturation (45 sec at 94°C., first cycle 4 min 45 sec), annealing (1 min at 65° C.), andextension (2 min at 72° C., last cycle 9 min) PCR reaction was subjectedto electrophoresis on a 3% Separide (Gibco) gel containing ethidiumbromide. The methylation-specific primers were FF280M (SEQ ID NO:231)(5′-TTTTTGCGTTTGTTGGAGAATCGGGTTTTC -3′) (forward) and FR432M (SEQ IDNO:232) (5′-ATACACCGCAAACCGCCGACGAACAAAACG-3′) (reverse). Theunmethylation-specific primers were FF280U (SEQ ID NO:233)(5′-TTTTTGTGTTTGTTGGAGAATTGGGTTTTT -3′) (forward) and FR432U (SEQ IDNO:234) (5′-ATACACCACAAACCACCAACAAACAAAACA -3′)(reverse). These primerswere designed using nt 280-309 for the forward primers and nt 403-432for the reverse primers.

TABLE 1 Complementation Groups and Responsible Genes of Fanconi AnemiaEstimated Number Sub- percentage Responsible Chromosome of Protein typeof patients gene location exons product A   66% FANCA 16q24.3 43    163Kd B  4.3% FANCB — — — C 12.7% FANCC 9q22.3 14    63 Kd D1 rare FANCD1 —— — D2 rare FANCD2 3p25.3 44 155,162 kD E 12.7% FANCE 6p21.2-21.3 10   60 kD F rare FANCF 11p15 1    42 kD G rare FANCG 9p13 14    68 kD(XRCC9)

TABLE 2 Diseases of Genomic Instability Disease Damaging Agent NeoplasmFunction FA Cross-linking agents Acute Unknown leukemia, hepatic,myeloblastic gastroinstestinal, and gynecological tumors XP UV lightSquamous cell Excision carcinomas repair AT Ionizing radiation LymphomaAfferent pathway to p53 Bloom's Alkylating agents Acute Cell-cycleSyndrome lymphoblastic regulation leukemia Cockayne's UV light Basalcell Transcription Syndrome carcinoma repair coupled Hereditary non-Unknown Adenocarcinoma DNA polyposis colon of colon, mismatch cancer(HNPCC) ovarian cancer repair

TABLE 3 FANCD2 Sequence Alterations Mutations PD20 nt376a→g S126G/splicent3707g→a R1236H VU008 nt904c→t R302W nt958c→t Q320X PD733 deletion ofexon 17 Polymorphisms nt1122a→g V374V nt1440t→c* H480H nt1509c→t^(†)N503N nt2141c→t*^(†) L714P nt2259t→c D753D nt4098t→g*^(†) L1366Lnt4453g→a^(†) 3UTR *PD20 is heterozygous; ^(†)VU008 is heterozygous.

TABLE 4 Chromosome Breakage Analysis of Whole-cell Fusions Cell DEB MMC% of Cells line/hybrids (ng/ml) (ng/ml) with radials Phenotype PD20i 30058 S PD24p 300 na* S VU423p 300 na* S PD319i 300 52 S PD20i/VU423p 300 6R PD20i/PD24p 300 30 S PD20i/PD319i 300 0 R PD20i 40 48 S VU423i 40 78 SPD20i/VU423i 40 10 R VU423i + chr. 3, 40 74 S clone 1 VU423i + chr. 3,40 68 S clone 2 VU423i + chr. 3, 40 88 S clone 3 PD20i + empty vector 00 2 40 24 S 200 62 S PD20i + FANCD2 vector 0 0 0 40 2 R 200 10 R Groupsof experiments are separated by line spaces. S, cross-linker sensitive;R, cross-linker-resistant; i = immortal fibroblast line; p = primaryfibroblasts. *Cell viability at this concentration was too low to scorefor radial formation, indicating the exquisite sensitivity of primaryfibroblasts to interstrand DNA-crosslinks.

TABLE 5 FA IR/ protein MMC Bleomycin FA complex sensitivity sensitivityCell line/plasmid Group (1) (2) (3) Lym- PD7 Wt + R R phoblasts HSC72 A− S HSC72 + A A + R PD4 C − S PD4 + C C + R EUFA316 G − S EUFA316 + GG + R EUFA121 F − S S EUFA121 + F F + R R PD20 D + S S PD20(R) D + R RFibro- GM0637 Wt + R R blasts GM6914 A − S S GM694 + A A + R R PD426 C −S PDF426 + C C + R FAG326SV G − S FAG326SV + G G + R PD20F D + S S20-3-15(+D) D + R R NBS (−/−) NBS + S S ATM (−/−) ATM + S S BRCA1 (−/−)BRCA1 + S S 1) The presence of the FA protein complex(FANCA/FANCG/FANCC) was determined as previously described(Garcia-Higuera et al., MCB 19:4866-4873, 1999) 2) MMC sensitivity fordetermined by the XTT assay for lymphoblasts or by the crystal violetassay for fibroblasts. 3) IR/Bleomycin sensitivity was determined byanalysis of chromosome breakage. (See Materials and Methods).

TABLE 6 The Intron/Exon Junctions of FANCD SEQ SEQ ID 5′-Donor ID3′-Acceptor Exon Size NO. site Score Intron NO. site Score Exon 1 30 9TCG 87 52 gtttcccgattttg 85 2 gtgagtaag ctctag GAA tg 2 97 10 CCA 83 53gaaaatttttctat 83 3 gtaagtact tttcag AAA cta 3 141 11 TAG 78 54ctcttcttttttctg 88 4 gtaatatttta catag CTG 4 68 12 AAA 81 159 55attttttaaatctcc 78 5 gtatgtatttt ttaag ATA 5 104 13 CAG 86 375 56gatttctttttttttt 91 6 gtgtggaga acag TAT gg 6 61 14 CAG 89 57ccctatgtcttctt 86 7 gtaagactg ttttag CCT tc 7 53 15 AAA 87 58ttctcttcctaaca 80 8 gtaagtggc ttttag CAA gt 8 79 16 AAG 83 364 59aatagtgtcttcta 85 9 gtaggcttatg ctgcag GAC 9 125 17 CAG 80 60tctttttctaccatt 86 10 gtggataaa cacag TGA cc 10 88 18 AAG 76 61tctgtgcttttaatt 85 11 gtagaaaag tttag GTT ac 11 105 19 GAG 80 387 62ctaatatttactttc 87 12 gtatgctctta tgcag GTA 12 101 20 AAG 85 342 63ttcctctctgctac 84 13 gtaaagagc ttgtag TTC tc 13 101 21 AAG 89 237 64actctctcctgttt 92 14 gtgagatcttt tttcag GCA 14 36 22 AAG 82 65tgcatatttattga 73 15 gtaatgttcat caatag GTG 15 144 23 TTA 80 66tctactcttcccc 86 16 gtaagtgtc actcaag GTT ag 16 135 24 CAG 85 67gttgactctcccc 84 17 gtatgttgaaa tgtatag GAA 17 132 25 AAG 77 68tggcatcatttttt 89 18 gtatcttattg ccacag GGC 18 111 26 CAG 83 69tcttcatcatctca 87 19 gttagaggc ttgcag GAT aa 19 110 27 CAG 82 70aaaaaattctttgt 79 20 gtacacgtg ttttag AAG ga 20 61 28 CAG 93 71attcttcctctttg 93 21 gtgagttcttt ctccag GTG 21 120 29 CTG 81 445 72tgtttgtttgcttcc 85 22 gtaaagcca tgaag GAA at 22 74 30 AGG 84 300 73attctggtttttctc 88 23 gtaggtattgt cgcag TGA 23 147 31 AAA 73 74aatttatttctcctt 89 24 gtcagtata ctcag ATT gt 24 101 32 TAG 84 370 75aaatgtttgttctc 86 25 gtatgggat tctcag ATT ga 25 116 33 GAG 88 76atgtaatttgtact 82 26 gtgagcag ttgcag ATT agt 26 109 34 CAG 89 77cagcctgctgttt 81 27 gtaagagaa gtttcag TCA gt 27 111 35 TAG 90 272 78ttctctttttaatat 73 29 gtaagtatgtt aaaag AAA 28 110 36 AAG 78 79ttgctgtgacttc 85 29 gtattggaatg cccatag GAG 29 144 37 GAA 85 80tcctttcctccatg 84 30 gtaagtgac tgacag GCT ag 30 117 38 AAG 86 81taactctgcattta 80 31 gttagtgtagg ttatagAAC 31 129 39 CAG 82 118 82aaaatcatttttatt 79 32 gtcagaagc tttag TGT ct 32 119 40 TTG 85 83tcttaccttgactt 85 33 gtaagtatgtg ccttag GAG 33 111 41 CAG 90 84tttttcttgtctcctt 91 34 gtgagtcat acag CCA aa 34 131 42 TTG 73 85tttgtcttcttttcta 89 35 gtgatgggc acag CTT ct 35 94 43 CTG 84 286 86atatttgactctca 78 36 gtgagatgttt atgcag TAT 36 123 44 CAG 92 87atgcttttcccgtc 88 37 gtaaggga ttctag GCA gtt 37 94 45 CAG 92 88catatatttggct 81 38 gtgagtaag gccccag at ATT 38 72 46 AAG 93 89cttgtctttcacct 93 39 gtgagtatg ctccag GTA ga 39 39 47 AAG 89 90agtgtgtctctctt 86 40 gtgagagat cttcag TAT tt 40 75 48 CGG 86 91tataaacttattgg 77 41 gtaagagct ttatag GAA aa 41 75 49 AAG 91 92tgttatttatttcca 86 42 gtaagaag ttcag ATT ggg 42 147 50 CAG 91 93cttggtccattca 80 43 gtaagcctt catttag GGT gg 43 228 CCA taa + 94attattctttgccc 44 3′UTR cttag GAT 96 51 GAG gtatctctaca 44 72 GAT tag +3′UTR

TABLE 7  PCR Primers to Amplify the 44 Exons of FANCD Primer SEQ IDProduct Annealing Exon Name NO. Primer Sequence (5′->3′) Size (bp) Temp 1 MG914 115 F: CTAGCACAGAACTCTGCTGC 372 54 MG837 116R: CTAGCACAGAACTCTGCTGC  2 MG746 117 F: CTTCAGCAACAGCGAAGTA- 422 50GTCTG MG747 118 R: ATTCTCAGCACTTGAAAAGC- AGG  3 MG773 119F: GGACACATCAGTTTTCCTCTC 309 50 MG789 120 R: GAAAACCCATGATTCAGTCC 4-5MG816 121 F: TCATCAGGCAAGAAACTTGG 467 50 MG803 122R: GAAGTTGGCAAAACAGACTG  6 MG804 123 F: GAGCCATCTGCTCATTTCTG 283 50MG812 124 R: CCCGCTATTTAGACTTGAGC  7 MG775 125 F: CAAAGTGTTTATTCCAGGAGC343 50 MG802 126 R: CATCAGGGTACTTTGAACA- TTC 8-9 MG727 127F: TTGACCAGAAAGGCTCAGT- 640 50 TCC MG915 128 R: AGATGATGCCAGAGGGTTTA-TCC 10 MG790 129 F: TGCCCAGCTCTGTTCAAACC 222 50 MG774 130R: AGGCAATGACTGACTGACAC 11 MG805 131 F: TGCCCGTCTATTTTTGATGA- 392 50 AGCMG791 132 R: TCTCAGTTAGTCTGGGGACAG 12 MG751 133 F: TCATGGTAGAGAGACTGGAC-432 50 TGTGC MG972 134 R: ACCCTGGAGCAAATGACAACC 13-14 MG973 135F: ATTTGCTCCAGGGTACATGGC 555 50 MG974 136 R: GAAAGACAGTGGGAAGGCA- AGC 15MG975 137 F: GGGAGTGTGTGGAACAAAT- 513 50 GAGC MG976 138R: AGTTTCTACAGGCTGGTCCT- ATTCC 16 MG755 139 F: AACGTGGAATCCCATTGATGC 37948 MG730 140 R: TTTCTGTGTTCCCTCCTTGC 17 MG794 141F: GATGGTCAAGTTACACTGGC 382 50 MG778 142 R: CACCTCCCACCAATTATAGT- ATTC18 MG808 143 F: CTATGTGTGTCTCTTTTACA- 234 48 GGG MG817 144R: AATCTTTCCCACCATATTGC 19 MG779 145 F: CATACCTTCTTTTGCTGTGC 199 48MG795 146 R: CCACAGAAGTCAGAATCTC- CACG 20 MG731 147F: TGTAACAAACCTGCACGTTG 632 56 MG732 148 R: TGCTACCCAAGCCAGTAGTT- TCC 21MG788 149 F: GAGTTTGGGAAAGATTGGC- 232 50 AGC MG772 150R: TGTAGTAAAGCAGCTCTCA- TGC 22-23 MG733 151 F: CAAGTACACTCTGCACTGCC 65250 MG758 152 R: TGACTCAACTTCCCCACCAA- GAG 24-25 MG736 153F: CTCCCTATGTACGTGGAGT- 732 50 AATAC MG737 154 R: GGGAGTCTTGTGGGAACTAAG26 MG780 155 F: TTCATAGACATCTCTCAGC- 284 50 TCTG MG759 156R: GTTTTGGTATCAGGGAAAGC 27-28 MG760 157 F: AGCCATGCTTGGAATTTTGG 653 50MG781 158 R: CTCACTGGGATGTCACAAAC 29 MG740 159 F: GGTCTTGATGTGTGACTTGT-447 50 ATCCC MG741 160 R: CCTCAGTGTCACAGTGTTCTT- TGTG 30 MG809 161F: CATGAAATGACTAGGACAT- 281 48 TCC MG797 162 R: CTACCCAGTGACCCAAACAC31-32 MG761 163 F: CGAACCCTTAGTTTCTGAGA- 503 50 CGC MG742 164R: TCAGTGCCTTGGTGACTGTC 33 MG916 165 F: TTGATGGTACAGACTGGAGGC 274 50MG810 166 R: AAGAAAGTTGCCAATCCTG- TTCC 34 MG762 167F: AGCACCTGAAAATAAGGAGG 343 50 MG743 168 R: GCCCAAAGTTTGTAAGTGT- GAG35-36 MG787 169 F: AGCAAGAATGAGGTCAAGTTC 590 50 MG806 170R: GGGAAAAACTGGAGGAAAG- AACTC 37 MG818 171 F: AGAGGTAGGGAAGGAAGCTAC 23350 MG813 172 R: CCAAAGTCCACTTCTTGAAG 38 MG834 173F: GATGCACTGGTTGCTACATC 275 50 MG836 174 R: CCAGGACACTTGGTTTCTGC 39MG839 175 F: ACACTCCCAGTTGGAATCAG 370 50 MG871 176R: CTTGTGGGCAAGAAATTGAG 40 MG829 177 F: TGGGCTGGATGAGACTATTC 223 50MG870 178 R: CCAAGGSVSYSYVYYVYHS- HVSSC 41 MG820 179F: TGATTATCAGCATAGGCTGG 271 50 MG811 180 R: GATCCCCCAATAGGAACTGC 42MG763 181 F: CATTCAGATTCACCAGGACAC 227 50 MG782 182R: CCTTACATGCCATCTGATGC 43 MG764 183 F: AACCTTCTCCCCTATTACCC 435 503′UTR MG835 184 R: GGAAAATGAGAGGCTATA- ATGC 44 MG1006 185F: TGTATTCCAGAGGTCACCC- 234 50 AGAGC 3′UTR MG1005 186R: CCAGTAAGAAAGGCAAACA- GCG

TABLE 8 FANCD2 LOCI on Human Chromosome 3p Copy Copy Copy Exon Copyregion 1 region 2 region 3 FANCD2 region 4 Copy region 5 1 201,110344,395 8,170,539 2 3 4 5 6 7 8 9 10 11 12 6,126,244 8,209,073 13 14 1516 8,202,791 17 18 19 20 21 22 23 24 18,201,854 25 26 27 28 6,094,448 2930 31 32 186,164 33 34 35 36 18,178,589 37 38 39 40 41 42 43 448,095,780

TABLE 9  Length of Primer Name Sequence Product SEQ ID NO:hFANCD2_super_1_2_R GGCCCACAGTTTCCGTTTCT — SEQ ID NO: 235hFANGD2_super_1_2_F CAAGGAAGCTAGAAATGAAGAAC SEQ ID NO: 236hFANCD2_super_3_3_R CTGGGACTACAGACACGTTTT — SEQ ID NO: 237hFANCD2_super_3_3_F GTGTCACGTGTCTGTAATCTC SEQ ID NO: 238hFANCD2_super_7_14_R TTAAGACCCAGCGAGGTATTC — SEQ ID NO: 239hFANCD2_super_7_14_F TGGGTTTGGTAGGGTAATGTC SEQ ID NO: 240hFANCD2_super_19_22_R TGGAAAGTCACTGCGGAGAAA — SEQ ID NO: 241hFANCD2_super_19_22_F ACGTAATCACCCCTGTAATCC SEQ ID NO: 242hFANGD2_super_23_29_R CACTGCAAACTGCTCACTCAA — SEQ ID NO: 243hFANCD2_super_23_29_F GGCCTTGTGCTAAGTGCTTTT SEQ ID NO: 244hFANCD2_super_30_32_R ACCCTGGTGGACATACCTTTT — SEQ ID NO: 245hFANCD2_super_30_32_F CCAAAGTACTGGGAGTTTGAG SEQ ID NO: 246hFANCD2_super_33_36_R TCTGGGCAACAGAACAAGCAA — SEQ ID NO: 247hFANCD2_super_33_36_F GAGCAATTTAGCCTGTGGTTTT SEQ ID NO: 248hFANCD2_super_43_44_R ACCATCTGGCCGACATGGTA — SEQ ID NO: 249hFANCD2_super_43_44_F AGGGTCCTGAGACTATATACC SEQ ID NO: 250hFANCD2_exon1_R TCCCATCTCAGGGCAGATGA 324 SEQ ID NO: 251 hFANCD2_exon1_FTATGCCCGGCTAGCACAGAA SEQ ID NO: 252 hFANCD2_exon2_RTCTCTCACATGCCTCACACAT 258 SEQ ID NO: 253 hFANCD2_exon2_FCCCCTCTGATTTTGGATAGAG SEQ ID NO: 254 hFANCD2_exon3_RAAGATGGATGGCCCTCTGATT 354 SEQ ID NO: 255 hFANCD2_exon3_FGACACATCAGTTTTCCTCTCAT SEQ ID NO: 256 hFANCD2_exon4_RAATCATTCTAGCCCACTCAACT 253 SEQ ID NO: 257 hFANCD2_exon4_FTGGTTTCATCAGGCAAGAAACT SEQ ID NO: 258 hFANCD2_exon5_RAGCCCCATGAAGTTGGCAAAA 298 SEQ ID NO: 259 hFANCD2_exon5_FGCTTGTGCCAGCATAACTCTA SEQ ID NO: 260 hFANCD2_exon6_RGCTGTGCTAAAGCTGCTACAA 341 SEQ ID NO: 261 hFANCD2_exon6_FGAGCCATCTGCTCATTTCTGT SEQ ID NO: 262 hFANCD2_exon7_RCAGAGAAACCAATAGTTTTCAG 280 SEQ ID NO: 263 hFANCD2_exon7_FAATCTCGGCTCACTGCAATCT SEQ ID NO: 264 hFANCD2_exon8_RAGCTAATGGATGGATGGAAAAG 333 SEQ ID NO: 265 hFANCD2_exon8_FTAGTGCAGTGCCGAATGCATA SEQ ID NO: 266 hFANCD2_exon9_RTACTCATGAAGGGGGGTATCA 323 SEQ ID NO: 267 hFANCD2_exon9_FTTCACACGTAGGTAGTCTTTCT SEQ ID NO: 268 hFANCD2_exon10_RCATTACTCCCAAGGCAATGAC 229 SEQ ID NO: 269 hFANCD2_exon10_FGCCCAGCTCTGTTCAAACCA SEQ ID NO: 270 hFANCD2_exon11_RAGCTCCATTCTCTCCTCTGAA 341 SEQ ID NO: 271 hFANCD2_exon11_FGTGGGAAGATGGAGTAAGAGA SEQ ID NO: 272 hFANCD2_exon12_RTCTGACAGTGGGATGTCAGAA 211 SEQ ID NO: 273 hFANCD2_exon12_FTGCCTACCCACTATGAATGAG SEQ ID NO: 274 hFANCD2_exon13_RATGTGTCCATCTGGCAACCAT 321 SEQ ID NO: 275 hFANCD2_exon13_FCAGGAACTCCGATCTTGTAAG SEQ ID NO: 276 hFANCD2_exon14_RTGGAGGGGGGAGAAAGAAAG 186 SEQ ID NO: 277 hFANCD2_exon14_FCGTGTTTCGCTGATGTGTCAT SEQ ID NO: 278 hFANCD2_exon15a_RGGAAGGCCAGTTTGTCAAAGT 325 SEQ ID NO: 279 hFANCD2_exon15a_FGTGTTTGACCTGGTGATGCTT SEQ ID NO: 280 hFANCD2_exon15b_RCTTATTTCTTAGCACCCTGTCAA 204 SEQ ID NO: 281 hFANCD2_exonl5b_FGTGGAACAAATGAGCATTATCC SEQ ID NO: 282 hFANCD2_exon16_RTTCCCCTTCAGTGAGTTCCAA 332 SEQ ID NO: 283 hFANCD2_exon16_FAGGGAGGAGAAGTCTGACATT SEQ ID NO: 284 hFANCD2_exon17_RGATTAGCCTGTAGGTTAGGTAT 422 SEQ ID NO: 285 hFANCD2_exon17_FGATGGGTTTGGGTTGATTGTG SEQ ID NO: 286 hFANCD2_exon18_RCCAGTCTAGGAGACAGAGCT 282 SEQ ID NO: 287 hFANCD2_exon18_FGGCTATCTATGTGTGTCTCTTT SEQ ID NO: 288 hFANCD2_exon19_RACGATTAGAAGGAACATGGAA 328 SEQ ID NO: 289 hFANCD2_exon19_FCGATATCCATACCTTCTTTTGC SEQ ID NO: 290 hFANCD2_exon20_RTGACAGAGCGAGACTCTCTAA 239 SEQ ID NO: 291 hFANCD2_exon20_FCACACCAACATGGCACATGTA SEQ ID NO: 292 hFANCD2_exon21_RGAGACAGGGTAGGGCAGAAA 339 SEQ ID NO: 293 hFANCD2_exon21_FAAAGGGGCGAGTGGAGTTTG SEQ ID NO: 294 hFANCD2_exon22_RGTAACTTCACCAGTGCAACCAA 279 SEQ ID NO: 295 hFANCD2_exon22_FATGCACTCTCTCTTTTCTACTT SEQ ID NO: 296 hFANCD2_exon23_RACAAGGAATCTGCCCCATTCT 356 SEQ ID NO: 297 hFANCD2_exon23_FTTCCCTGTAGCCTTGCGTATT SEQ ID NO: 298 hFANCD2_exon24_RCCCCACATACACCATGTATTG 258 SEQ ID NO: 299 hFANCD2_exon24_FCTCCCTATGTACGTGGAGTAA SEQ ID NO: 300 hFANCD2_exon25_RGTGGGACATAACAGCTAGAGA 350 SEQ ID NO: 301 hFANCD2_exon25_FAGGGGAAAGTAAATAGCAAGGA SEQ ID NO: 302 hFANCD2_exon26_RTCAGGGATATTGGCCTGAGAT 324 SEQ ID NO: 303 hFANCD2_exon26_FGACATCTCTCAGCTCTGGATA SEQ ID NO: 304 hFANCD2_exon27_RCCAATTACTGATGCCATGATAC 324 SEQ ID NO: 305 hFANCD2_exon27_FGCATTCAGCCATGCTTGGTAA SEQ ID NO: 306 hFANCD2_exon28_RGATTACTCCAACGCCTAAGAG 354 SEQ ID NO: 307 hFANCD2_exon28_FTCTACCTCTAGGCAGTTTCCA SEQ ID NO: 308 hFANCD2_exon29_RTCTCCTCAGTGTCACAGTCTT 384 SEQ ID NO: 309 hFANCD2_exon29_FCTTGGGCTAGAGGAAGTTGTT SEQ ID NO: 310 hFANCD2_exon30_RTACCCAGTGACCCAAACACAA 348 SEQ ID NO: 311 hFANCD2_exon30_FGAGTTCAAGGCTGGAATAGCT SEQ ID NO: 312 hFANCD2_exon31_RACCGTGATTCTCACCAGCTAA 341 SEQ ID NO: 313 hFANCD2_exon31_FCCATTGCGAACCCTTAGTTTC SEQ ID NO: 314 hFANCD2_exon32_RAGTGCTTGGTGACTGTCAAA 336 SEQ ID NO: 315 hFANCD2_exon32_FCCACCTGGAGAACATTCACAA SEQ ID NO: 316 hFANCD2_exon33_RTACTGAAAGACACCCAGGTTAT 340 SEQ ID NO: 317 hFANCD2_exon33_FCACGCCCGACCTCTCAATTC SEQ ID NO: 318 hFANCD2_exon34_RTATAGCAAGAGGGCCTATCCA 349 SEQ ID NO: 319 hFANCD2_exon34_FTTGGGCACGTCATGTGGATTT SEQ ID NO: 320 hFANCD2_exon35_RGTCCAGTCTCTGACAAACAAC 100 SEQ ID NO: 321 hFANCD2_exon35_FTTAGACCGGGAACGTCTTAGT SEQ ID NO: 322 hFANCD2_exon36_RGGCCAAGTGGGTCTCAAAAC 398 SEQ ID NO: 323 hFANCD2_exon36_FCCTCTGGTTCTGTTTTATACTG SEQ ID NO: 324 hFANCD2_exon37_RTCTGGGCAACGAACAAGCAA 277 SEQ ID NO: 325 hFANCD2_exon37_FCTTCCCAGGTAGTTCTAAGCA SEQ ID NO: 326 hFANCD2_exon38_RAAGCCAGGACACTTGGTTTCT 274 SEQ ID NO: 327 hFANCD2_exon38_FGCACTGGTTGCTACATCTAAG SEQ ID NO: 328 hFANCD2_exon39_RGCATCCATTGCCTTCCCTAAA 236 SEQ ID NO: 329 hFANCD2_exon39_FTGCTCAAAGGAGCAGATCTCA SEQ ID NO: 330 hFANCD2_exon40_RCAGTCCAATTTGGGGATCTCT 309 SEQ ID NO: 331 hFANCD2_exon40_FCCTTGGGCTGGATGAGACTA SEQ ID NO: 332 hFANCD2_exon41_RCCCCAATAGCAACTGCAGATT 214 SEQ ID NO: 333 hFANCD2_exon41_FGATTGCAAGGGTATCTTGAATC SEQ ID NO: 334 hFANCD2_exon42_RGCTTAGGTGACCTTCCTTACA 356 SEQ ID NO: 335 hFANCD2_exon42_FAACATACCGTTGGCCCATACT SEQ ID NO: 336 hFANCD2_exon43a_RAGCATGATCTCGGCTCACCA 366 SEQ ID NO: 337 hFANCD2_exon43a_FGTGGCTCATGCTTGTAATCCT SEQ ID NO: 338 hFANCD2_exon43b_RTCAGTAGAGATGGGGTTTCAC 358 SEQ ID NO: 339 hFANCD2_exon43b_FCTGCCACCTTAGAGAACTGAA SEQ ID NO: 340 hFANCD2_exon43c_RCTCAAGCAATCCTCCTACCTT 405 SEQ ID NO: 341 hFANCD2_exon43c_FTAGAATCACTCCTGAGTATCTC SEQ ID NO: 342 hFANCD2_exon43d_RCAGCTTCTGACTCTGTGCTTT 367 SEQ ID NO: 343 hFANCD2_exon43d_FAGTTGGTGGAGCAGAACTTTG SEQ ID NO: 344 hFANCD2_exon43e_RCTCGAGATACTCAGGAGTGAT 381 SEQ ID NO: 345 hFANCD2_exon43e_FTCAACCTTCTCCCCTATTACC SEQ ID NO: 346 hFANCD2_exon43f_RAGTTCTGCTCCACCAACTTAG 306 SEQ ID NO: 347 hFANCD2_exon43f_FGGTATCCATGTTTGCTGTGTTT SEQ ID NO: 348 hFANCD2_exon44_RGAAAGGCAAACAGCGGATTTC 213 SEQ ID NO: 349 hFANCD2_exon44_FCACCCAGAGCAGTAACCTAAA SEQ ID NO: 350

All patents, patent application, and published references cited hereinare hereby incorporated by reference in their entirety. While thisinvention has been particularly shown and described with references topreferred embodiments thereof, it will be understood by those skilled inthe art that various changes in form and details may be made thereinwithout departing from the scope of the invention encompassed by theappended claims.

1.-41. (canceled)
 42. A method of determining the response of a cellfrom a subject to an agent capable of inducing DNA damage, comprising:a) providing a tissue sample from said subject; b) inducing DNA damagein the cells of said tissue sample; and c) detecting FANCD2ubiquitination or FANCD2 foci formation in said cells, wherein a lowlevel of FANCD2 ubiquitination or FANCD2 foci formation detected in c)as compared to the level of FANCD2 ubiquitination or FANCD2 fociformation in a control cell indicates that said cell will respond tosaid agent.
 43. The method of claim 42, wherein said cell is a cancercell.
 44. The method of claim 43, wherein said cancer is breast cancer,ovarian cancer, or prostate cancer.
 45. The method of claim 42, whereinDNA damage is induced in said control cell before FANCD2 ubiquitinationor FANCD2 foci formation is detected in said control cell.
 46. Themethod of claim 42, wherein said DNA damage in b) is induced by an agentselected from the group consisting of ethidium bromide, acridine orange,free radicals, ionizing radiation, and UV radiation.
 47. The method ofclaim 42, wherein said detection in c) comprises an immunologicalmethod.
 48. The method of claim 47, wherein said immunological method isimmunohistochemistry, immunofluorescence, or immunoblotting.
 49. Themethod of claim 42, wherein said agent is cisplatin.
 50. The method ofclaim 43, wherein said ubiquitination is monoubiquitination.
 51. Amethod of detecting a ubiquitinated FANCD2, comprising: a) providing oneor more cells from a subject; b) inducing DNA damage in said cells; andc) detecting said ubiquitinated FANCD2 in said cells by an immunologicalassay.
 52. The method of claim 51, wherein said immunological assay isimmunohistochemistry, immunofluorescence, or immunoblotting.
 53. Themethod of claim 51, wherein said ubiquitinated FANCD2 is amonoubiquitinated.